Mediators of Hedgehog Signaling Pathways, Compositions and Uses Related Thereto

ABSTRACT

The present invention makes available methods and reagents for inhibiting aberrant growth states resulting from hedgehog gain-of-function, ptc loss-of-function or smoothened gain-of-function comprising contacting the cell with a hedgehog antagonist, such as a small molecule, in a sufficient amount to aberrant growth state, e.g., to agonize a normal ptc pathway or antagonize smoothened or hedgehog activity.

This application claims priority to U.S. Provisional Application No.60/624,536, filed Nov. 3, 2004, the specification of which is herebyincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Hedgehog (Hh) protein was first identified in Drosophila melanogaster asa segment-polarity gene involved in embryo patterning (Nusslein-Volhardet al., Roux. Arch. Dev. Biol. 193: 267-282 (1984)). Three orthologs ofDrosophila hedgehog (Sonic, Desert and Indian) were later identified tooccur in all vertebrates including fish, birds and mammals. Deserthedgehog (DHh) is expressed principally in the testes, both in mouseembryonic development and in the adult rodent and human; Indian hedgehog(IHh) is involved in bone development during embryogenesis and in boneformation in the adult; and, Sonic hedgehog (SHh) is expressed at highlevels in the notochord and floor plate of developing vertebrateembryos. In vitro explant assays as well as ectopic expression of SHh intransgenic animals have shown that SHh plays a key role in neuronal tubepatterning (Echelard et al., supra; Ericson et al., Cell 81: 747-56(1995); Marti et al., Nature 375: 322-5 (1995); Krauss et al., Cell 75,1432-44 (1993); Riddle et al., Cell 75: 1401-16 (1993); Roelink et al,Cell 81:445-55 (1995); Hynes et al., Neuron 19: 15-26 (1997)). Hh alsoplays a role in the development of limbs (Krauss et al., Cell 75:1431-44 (1993); Laufer et al., Cell 79, 993-1003 (1994)), somites (Fanand Tessier-Lavigne, Cell 79, 1175-86 (1994); Johnson et al., Cell 79:1165-73 (1994)), lungs (Bellusci et al., Develop. 124: 53-63 (1997) andskin (Oro et al., Science 276: 817-21 (1997)). Likewise, IHh and DHh areinvolved in bone, gut and germinal cell development (Apelqvist et al.,Curr. Biol. 7: 801-4 (1997); Bellusci et al., Dev. Suppl. 124: 53-63(1997); Bitgood et al., Curr. Biol. 6: 298-304 (1996); Roberts et al.,Development 121: 3163-74 (1995)).

Human SHh is synthesized as a 45 kDa precursor protein that uponautocatalytic cleavage yields a 20 kDa N-terminal fragment that isresponsible for normal hedgehog signaling activity; and a 25 kDaC-terminal fragment that is responsible for autoprocessing activity inwhich the N-terminal fragment is conjugated to a cholesterol moiety(Lee, J. J., et al. (1994) Science 266, 1528-1536; Bumcrot, D. A., etal. (1995), Mol. Cell. Biol. 15, 2294-2303; Porter, J. A., et al. (1995)Nature 374, 363-366). The N-terminal fragment consists of amino acidresidues 24-197 of the full-length precursor sequence which remainsmembrane-associated through the cholesterol at its C-terminus (Porter,J. A., et al. (1996) Science 274, 255-258; Porter, J. A., et al. (1995)Cell 86, 21-34). Cholesterol conjugation is responsible for the tissuelocalization of the hedgehog signal.

At the cell surface, the Hh signal is thought to be relayed by the 12transmembrane domain protein Patched (Ptc) (Hooper and Scoff, Cell 59:751-65 (1989); Nakano et al., Nature 341: 508-13 (1989)) and theG-protein-coupled-like receptor Smoothened (Smo) (Alcedo et al., Cell86: 221-232 (1996); van den Heuvel and Ingham, Nature 382: 547-551(1996)). Both genetic and biochemical evidence support a receptor modelwhere Ptc and Smo are part of a multicomponent receptor complex (Chenand Struhl, Cell 87: 553-63 (1996); Marigo et al., Nature 384: 176-9(1996); Stone et al., Nature 384: 129-34 (1996)). Upon binding of Hh toPtc, the normal inhibitory effect of Ptc on Smo is relieved, allowingSmo to transduce the Hh signal across the plasma membrane. However, theexact mechanism by which Ptc controls Smo activity still has yet to beclarified.

The signaling cascade initiated by Smo results in activation of Glitranscription factors that translocate into the nucleus where theycontrol transcription of target genes. Gli has been shown to influencetranscription of Hh pathway inhibitors such as Ptc and Hip1 in anegative feedback loop indicating that tight control the Hh pathwayactivity is required for proper cellular differentiation and organformation. Uncontrolled activation of Hh signaling pathway areassociated with malignancies in particular those of the brain, skin andmuscle as well as angiogenesis. An explanation for this is that Hhpathway has been shown to regulate cell proliferation in adults byactivation of genes involved in cell cycle progression such as cyclin Dwhich is involved in G1-S transition. Also, SHh blocks cell-cycle arrestmediated by p21, an inhibitor of cyclin dependent kinases. Hh signalingis further implicated in cancer by inducing components in the EGFRpathway (EGF, Her2) involved in proliferation as well as components inthe PDGF (PDGFoe) and VEGF pathways involved in angiogenesis. Loss offunction mutations in the Ptc gene have been identified in patients withthe basal cell nevus syndrome (BCNS), a hereditary disease characterizedby multiple basal cell carcinomas (BCCs). Dysfunctional Ptc genemutations have also been associated with a large percentage of sporadicbasal cell carcinoma tumors (Chidambaram et al., Cancer Research 56:4599-601 (1996); Gailani et al., Nature Genet. 14: 78-81 (1996); Hahn etal., Cell 85: 841-51 (1996); Johnson et al., Science 272: 1668-71(1996); Unden et al., Cancer Res. 56: 4562-5; Wicking et al., Am. J.Hum. Genet. 60: 21-6 (1997)). Loss of Ptc function is thought to causean uncontrolled Smo signaling in basal cell carcinoma. Similarly,activating Smo mutations have been identified in sporadic BCC tumors(Xie et al., Nature 391: 90-2 (1998)), emphasizing the role of Smo asthe signaling subunit in the receptor complex for SHh.

Various inhibitors of hedgehog signaling have been investigated such ascyclopamine, a natural alkaloid that has been shown to arrest cell cycleat G0-G1 and to induce apoptosis in SCLC. Cyclopamine is believed toinhibit Smo by binding to its heptahelical bundle. Forskolin has beenshown to inhibit the Hh pathway downstream from Smo by activatingprotein kinase A (PKA) which maintains Gli transcription factorsinactive. Despite advances with these and other compounds, there remainsa need for potent inhibitors of the hedgehog signaling pathway.

SUMMARY OF THE INVENTION

The present invention makes available methods and reagents forinhibiting activation of the hedgehog signaling pathway, e.g., toinhibit aberrant growth states resulting from phenotypes such as ptcloss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function, comprising contacting the cell with an agent, such asa small molecule, in a sufficient amount to agonize a normal ptcactivity, antagonize a normal hedgehog activity, or antagonizesmoothened activity, e.g., to reverse or control the aberrant growthstate.

One embodiment of the present invention provides a compound representedin the general formula (I):

wherein, as valence and stability permit,

-   -   X and Z, independently, represent —N(R₇)—, —O—, —S—,        —(R₇)N—N(R₇)—, —ON(R₇)—, or a direct bond;

Y represents —C(═O)—, —C(═S)—, —C(═NR₇)—, SO₂, or SO;

A represents O, S, or NR₇;

G represents a cycloalkyl, heterocyclyl, aryl, or heteroaryl ring fusedto the ring to which it is attached;

Ar represents a substituted or unsubstituted aryl or heteroaryl ring;

R₁ represents a disubstituted pyridine ring;

R₂ represents from 0-4 substituents on the ring to which it is attached;

R₇, independently for each occurrence, represents H, lower alkyl,J-cycloalkyl, J-heterocyclyl, J-aryl, J-heteroaryl;

R₈, independently for each occurrence, represents H, lower alkyl,cycloalkyl, heterocyclyl, aryl, or heteroaryl; and

J represents, independently for each occurrence, a chain having from 0-8units selected from CK₂, NK, O, and S, wherein K represents,independently for each occurrence, H or lower alkyl.

In certain embodiments, at least one of Z and X is not a direct bond.

In certain embodiments, X-Y-Z includes an amide, urea, or sulfonamide.

In certain embodiments, X is selected from —N(R₈)—, —O—, —S—, andpreferably represents NH.

In certain embodiments, R₁ is substituted with a methyl ortrifluoromethyl group, e.g., 6-(trifluoromethyl)-2-methylpyridin-3-yl.

In certain embodiments, X and the ring comprising A are disposed on Arin a meta relationship.

In certain embodiments, R₂ represents from 1-4 substituents selectedfrom halogen, lower alkyl, lower alkenyl, aryl, heteroaryl, carbonylgroup, thiocarbonyl, ketone, aldehyde, amino, acylamino, amido, amidino,cyano, nitro, azido, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl,sulfonamido, phosphoryl, phosphonate, phosphinate, J-R₈, J-OH, J-loweralkyl, J-lower alkenyl, J-R₈, J-SH, J-NH₂, protected forms of the above,or any two R₂, when occurring more than once in a cyclic or polycyclicstructure, can be taken together form a 4- to 8-membered cycloalkyl,aryl, or heteroaryl.

In certain embodiments, R₂ represents from 1-4 substituents selectedfrom halogen, cyano, nitro, alkoxy, amino, acylamino (e.g.,R₈—C(═O)NH—), a substituted or unsubstituted cycloalkyl, heterocyclyl,aryl, or heteroaryl fused to G, and substituted or unsubstituted loweralkyl.

Another embodiment of the present invention provides a method forinhibiting activation of a hedgehog pathway in a cell, comprisingcontacting the cell with a compound as described above in a sufficientamount to inhibit hedgehog signalling.

In certain such embodiments, the cell has a phenotype of ptcloss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function.

In certain such embodiments, the compound inhibits ptc loss-of-function,hedgehog gain-of-function, or smoothened gain-of-function mediatedsignal transduction with an ED₅₀ of 1 μM or less, or even 1 nM or less.

In certain embodiments, the cell is contacted with the hedgehogantagonist in vitro or in vivo.

In certain embodiments, the compound is administered as part of atherapeutic or cosmetic application, such as regulation of neuraltissues, bone and cartilage formation and repair, regulation ofspermatogenesis, regulation of smooth muscle, regulation of lung, liverand other organs arising from the primative gut, regulation ofhematopoietic function, and regulation of skin and hair growth.

Another embodiment of the invention provides a pharmaceuticalpreparation comprising a sterile pharmaceutical excipient and a compoundas described above.

Another embodiment of the invention provides a method for treating orpreventing basal cell carcinoma, comprising administering a compound orpharmaceutical preparation as described above to a patient in an amountsufficient to inhibit progression of basal cell carcinoma.

Another embodiment of the invention provides a compound represented inthe general formula (I):

wherein, as valence and stability permit,

X and Z, independently, represent —N(R₇)—, —O—, —S—, —(R₇)N—N(R₇)—,—ON(R₇)—, or a direct bond;

Y represents —C(═O)—, —C(═S)—, —C(═NR₇)—, SO₂, or SO;

A represents O, S, or NR₇;

G represents a cycloalkyl, heterocyclyl, aryl, or heteroaryl ring fusedto the ring to which it is attached;

Ar represents a substituted or unsubstituted aryl or heteroaryl ring;

R₁ represents a pyridine ring substituted with one or more substituents,wherein at least one of the substituents is selected from the groupnitro, cyano, lower alkyl, halogenated lower alkyl, alkenyl, alkynyl,amino, alkylamino, acylamino, amido, hydroxyl, alkoxy, acyloxy,carbonyl, sulfhydryl, or sulfonyl;

R₂ represents from 0-4 substituents on the ring to which it is attached;

R₇, independently for each occurrence, represents H, lower alkyl,J-cycloalkyl, J-heterocyclyl, J-aryl, J-heteroaryl;

R₈, independently for each occurrence, represents H, lower alkyl,cycloalkyl, heterocyclyl, aryl, or heteroaryl; and

J represents, independently for each occurrence, a chain having from 0-8units selected from CK₂, NK, O, and S, wherein K represents,independently for each occurrence, H or lower alkyl.

In certain embodiments, at least one of Z and X is not a direct bond. Incertain embodiments, X-Y-Z includes an amide, urea, or sulfonamide.

In certain embodiments, X is selected from —N(R₈)—, —O—, —S—, andpreferably represents NH.

In certain embodiments, R₁ is substituted with a methyl ortrifluoromethyl group, e.g., R₁ is6-(trifluoromethyl)-2-methylpyridin-3-yl.

In certain embodiments, X and the ring comprising A are disposed on Arin a meta relationship.

In certain embodiments, R₂ represents from 1-4 substituents selectedfrom halogen, lower alkyl, lower alkenyl, aryl, heteroaryl, carbonylgroup, thiocarbonyl, ketone, aldehyde, amino, acylamino, amido, amidino,cyano, nitro, azido, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl,sulfonamido, phosphoryl, phosphonate, phosphinate, J-R₈, J-OH, J-loweralkyl, J-lower alkenyl, J-R₉, J-SH, J-NH₂, protected forms of the above,or any two R₂, when occurring more than once in a cyclic or polycyclicstructure, can be taken together form a 4- to 8-membered cycloalkyl,aryl, or heteroaryl.

In certain embodiments, R₂ represents from 1-4 substituents selectedfrom halogen, cyano, nitro, alkoxy, amino, acylamino (e.g.,R₈—C(═O)NH—), a substituted or unsubstituted cycloalkyl, heterocyclyl,aryl, or heteroaryl fused to G, and substituted or unsubstituted loweralkyl.

Another embodiment of the present invention provides a method forinhibiting activation of a hedgehog pathway in a cell, comprisingcontacting the cell with a compound as described above in a sufficientamount to inhibit hedgehog signalling,

In certain such embodiments, the cell has a phenotype of ptcloss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function.

In certain such embodiments, the compound inhibits ptc loss-of-function,hedgehog gain-of-function, or smoothened gain-of-function mediatedsignal transduction with an ED₅₀ of 1 μM or less, or even 1 nM or less.

In certain embodiments, the cell is contacted with the hedgehogantagonist in vitro or in vivo.

In certain embodiments, the compound is administered as part of atherapeutic or cosmetic application, such as regulation of neuraltissues, bone and cartilage formation and repair, regulation ofspermatogenesis, regulation of smooth muscle, regulation of lung, liverand other organs arising from the primative gut, regulation ofhematopoietic function, and regulation of skin and hair growth.

Another embodiment of the present invention provides a pharmaceuticalpreparation comprising a sterile pharmaceutical excipient and a compoundas described above.

Another embodiment of the present invention provides a method fortreating or preventing basal cell carcinoma, comprising administering acompound or pharmaceutical preparation as described above to a patientin an amount sufficient to inhibit progression of basal cell carcinoma.

Another embodiment of the present invention provides a compoundrepresented in the general formula (I):

wherein, as valence and stability permit,

X and Z, independently, represent —N(R₇)—, —O—, —S—, —(R₇)N—N(R₇)—,—ON(R₇)—, or a direct bond;

Y represents —C(═O)—, —C(═S)—, —C(═NR₇)—, SO₂, or SO;

A represents O, S, or NR₇;

G represents a cycloalkyl, heterocyclyl, aryl, or heteroaryl ring fusedto the ring to which it is attached;

Ar represents a substituted or unsubstituted aryl or heteroaryl ring;

R₁ represents a substituted pyridine ring attached to J at a positionmeta to the nitrogen atom;

R₂ represents from 0-4 substituents on the ring to which it is attached;

R₇, independently for each occurrence, represents H, lower alkyl,J-cycloalkyl, J-heterocyclyl, J-aryl, J-heteroaryl;

R₈, independently for each occurrence, represents H, lower alkyl,cycloalkyl, heterocyclyl, aryl, or heteroaryl; and

J represents, independently for each occurrence, a chain having from 0-8units selected from CK₂, NK, O, and S, wherein K represents,independently for each occurrence, H or lower alkyl.

In certain embodiments, at least one of Z and X is not a direct bond.

In certain embodiments, X-Y-Z includes an amide, urea, or sulfonamide.

In certain embodiments, X is selected from —N(R₈)—, —O—, —S—, andpreferably represents NH.

In certain embodiments, R₁ is substituted with a methyl ortrifluoromethyl group.

In certain embodiments, R₁ is 6-(trifluoromethyl)-2-methylpyridin-3-yl.

In certain embodiments, X and the ring comprising A are disposed on Arin a meta relationship.

In certain embodiments, R₂ represents from 1-4 substituents selectedfrom halogen, lower alkyl, lower alkenyl, aryl, heteroaryl, carbonylgroup, thiocarbonyl, ketone, aldehyde, amino, acylamino, amido, amidino,cyano, nitro, azido, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl,sulfonamido, phosphoryl, phosphonate, phosphinate, J-R₈, J-OH, J-loweralkyl, J-lower alkenyl, J-R₉, J-SH, J-NH₂, protected forms of the above,or any two R₂, when occurring more than once in a cyclic or polycyclicstructure, can be taken together form a 4- to 8-membered cycloalkyl,aryl, or heteroaryl.

In certain embodiments, R₂ represents from 1-4 substituents selectedfrom halogen, cyano, nitro, alkoxy, amino, acylamino (e.g.,R₉—C(═O)NH—), a substituted or unsubstituted cycloalkyl, heterocyclyl,aryl, or heteroaryl fused to G, and substituted or unsubstituted loweralkyl.

Another embodiment of the present invention provides a method forinhibiting activation of a hedgehog pathway in a cell, comprisingcontacting the cell with a compound as described above in a sufficientamount to inhibit hedgehog signalling,

In certain such embodiments, the cell has a phenotype of ptcloss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function.

In certain such embodiments, the compound inhibits ptc loss-of-function,hedgehog gain-of-function, or smoothened gain-of-function mediatedsignal transduction with an ED₅₀ of 1 μM or less, or even 1 nM or less.

In certain embodiments, the cell is contacted with the hedgehogantagonist in vitro or in vivo.

In certain embodiments, the compound is administered as part of atherapeutic or cosmetic application, such as regulation of neuraltissues, bone and cartilage formation and repair, regulation ofspermatogenesis, regulation of smooth muscle, regulation of lung, liverand other organs arising from the primative gut, regulation ofhematopoietic function, and regulation of skin and hair growth.

Another embodiment of the present invention provides a pharmaceuticalpreparation comprising a sterile pharmaceutical excipient and thecompound above.

Another embodiment of the present invention provides a method fortreating or preventing basal cell carcinoma, comprising administering acompound or pharmaceutical preparation as described above to a patientin an amount sufficient to inhibit progression of basal cell carcinoma.

DETAILED DESCRIPTION OF THE INVENTION I. Overview

The present invention relates to the discovery that signal transductionpathways regulated by hedgehog, patched (ptc), gli and/or smoothened canbe inhibited, at least in part, by small molecules. While not wishing tobe bound by any particular theory, the activation of a receptor may bethe mechanism by which these agents act. For example, the ability ofthese agents to inhibit proliferation of patched loss-of-function(ptc^(lof)) cells may be due to the ability of such molecules tointeract with hedgehog, patched, or smoothened, or at least to interferewith the ability of those proteins to activate a hedgehog, ptc, and/orsmoothened-mediated signal transduction pathway.

It is, therefore, specifically contemplated that these small moleculeswhich intefere with aspects of hedgehog, ptc, or smoothened signaltransduction activity will likewise be capable of inhibitingproliferation (or other biological consequences) in normal cells and/orcells having a patched loss-of-function phenotype, a hedgehoggain-of-function phenotype, or a smoothened gain-of-function phenotype.Thus, it is contemplated that in certain embodiments, these compoundsmay be useful for inhibiting hedgehog activity in normal cells, e.g.,which do not have a genetic mutation that activates the hedgehogpathway. In preferred embodiments, the subject inhibitors are organicmolecules having a molecular weight less than 2500 amu, more preferablyless than 1500 amu, and even more preferably less than 750 amu, and arecapable of inhibiting at least some of the biological activities ofhedgehog proteins, preferably specifically in target cells.

Thus, the methods of the present invention include the use of smallmolecules which agonize ptc inhibition of hedgehog signalling, such asby inhibiting activation of smoothened or downstream components of thesignal pathway, in the regulation of repair and/or functionalperformance of a wide range of cells, tissues and organs, includingnormal cells, tissues, and organs, as well as those having the phenotypeof ptc loss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function. For instance, the subject method has therapeutic andcosmetic applications ranging from regulation of neural tissues, boneand cartilage formation and repair, regulation of spermatogenesis,regulation of smooth muscle, regulation of lung, liver and other organsarising from the primative gut, regulation of hematopoietic function,regulation of skin and hair growth, etc. Moreover, the subject methodscan be performed on cells that are provided in culture (in vitro), or oncells in a whole animal (in vivo). See, for example, PCT publications WO95/18856 and WO 96/17924 (the specifications of which are expresslyincorporated by reference herein).

In a preferred embodiment, the subject method can be to treat epithelialcells having a phenotype of ptc loss-of-function, hedgehoggain-of-function, or smoothened gain-of-function. For instance, thesubject method can be used in treating or preventing basal cellcarcinoma or other hedgehog pathway-related disorders.

In certain embodiments, a subject antagonist may inhibit activation of ahedgehog pathway by binding to smoothened. In certain embodiments, asubject antagonist may inhibit activation of a hedgehog pathway by bbinding to patched.

In another preferred embodiment, the subject method can be used as partof a treatment regimen for malignant medulloblastoma and other primaryCNS malignant neuroectodermal tumors.

In another aspect, the present invention provides pharmaceuticalpreparations comprising, as an active ingredient, a hedgehog antagonist,ptc agonist, or smoothened antagonist such as described herein,formulated in an amount sufficient to inhibit, in vivo, proliferation orother biological consequences of ptc loss-of-function, hedgehoggain-of-function, or smoothened gain-of-function.

The subject treatments using hedgehog antagonists, patched agonists, orsmoothened antagonists can be effective for both human and animalsubjects. Animal subjects to which the invention is applicable extend toboth domestic animals and livestock, raised either as pets or forcommercial purposes. Examples are dogs, cats, cattle, horses, sheep,hogs, and goats.

II. Definitions

For convience, certain terms employed in the specification, examples,and appended claims are collected here.

The phrase “aberrant modification or mutation” of a gene refers to suchgenetic lesions as, for example, deletions, substitution or addition ofnucleotides to a gene, as well as gross chromosomal rearrangements ofthe gene and/or abnormal methylation of the gene. Likewise,mis-expression of a gene refers to aberrant levels of transcription ofthe gene relative to those levels in a normal cell under similarconditions, as well as non-wild-type splicing of mRNA transcribed fromthe gene.

“Basal cell carcinomas” exist in a variety of clinical and histologicalforms such as nodular-ulcerative, superficial, pigmented, morphealike,fibroepithelioma and nevoid syndrome. Basal cell carcinomas are the mostcommon cutaneous neoplasms found in humans. The majority of new cases ofnonmelanoma skin cancers fall into this category.

“Burn wounds” refer to cases where large surface areas of skin have beenremoved or lost from an individual due to heat and/or chemical agents.

The term “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate surrounding tissues and to giverise to metastasis. Exemplary carcinomas include: “basal cellcarcinoma”, which is an epithelial tumor of the skin that, while seldommetastasizing, has potentialities for local invasion and destruction;“squamous cell carcinoma”, which refers to carcinomas arising fromsquamous epithelium and having cuboid cells; “carcinosarcoma”, whichinclude malignant tumors composed of carcinomatous and sarcomatoustissues; “adenocystic carcinoma”, carcinoma marked by cylinders or bandsof hyaline or mucinous stroma separated or surrounded by nests or cordsof small epithelial cells, occurring in the mammary and salivary glands,and mucous glands of the respiratory tract; “epidermoid carcinoma”,which refers to cancerous cells which tend to differentiate in the sameway as those of the epidermis; i.e., they tend to form prickle cells andundergo cornification; “nasopharyngeal carcinoma”, which refers to amalignant tumor arising in the epithelial lining of the space behind thenose; and “renal cell carcinoma”, which pertains to carcinoma of therenal parenchyma composed of tubular cells in varying arrangements.Other carcinomatous epithelial growths are “papillomas”, which refers tobenign tumors derived from epithelium and having a papillomavirus as acausative agent; and “epidermoidomas”, which refers to a cerebral ormeningeal tumor formed by inclusion of ectodermal elements at the timeof closure of the neural groove.

The “corium” or “dermis” refers to the layer of the skin deep to theepidermis, consisting of a dense bed of vascular connective tissue, andcontaining the nerves and terminal organs of sensation. The hair roots,and sebaceous and sweat glands are structures of the epidermis which aredeeply embedded in the dermis.

“Dental tissue” refers to tissue in the mouth that is similar toepithelial tissue, for example gum tissue. The method of the presentinvention is useful for treating periodontal disease.

“Dermal skin ulcers” refer to lesions on the skin caused by superficialloss of tissue, usually with inflammation. Dermal skin ulcers that canbe treated by the method of the present invention include decubitusulcers, diabetic ulcers, venous stasis ulcers and arterial ulcers.Decubitus wounds refer to chronic ulcers that result from pressureapplied to areas of the skin for extended periods of time. Wounds ofthis type are often called bedsores or pressure sores. Venous stasisulcers result from the stagnation of blood or other fluids fromdefective veins. Arterial ulcers refer to necrotic skin in the areaaround arteries having poor blood flow.

The term “ED₅₀” means the dose of a drug that produces 50% of itsmaximum response or effect.

An “effective amount” of, e.g., a hedgehog antagonist, with respect tothe subject method of treatment, refers to an amount of the antagonistin a preparation which, when applied as part of a desired dosage regimenbrings about, e.g., a change in the rate of cell proliferation and/orthe state of differentiation of a cell and/or rate of survival of a cellaccording to clinically acceptable standards for the disorder to betreated or the cosmetic purpose.

The terms “epithelia”, “epithelial” and “epithelium” refer to thecellular covering of internal and external body surfaces (cutaneous,mucous and serous), including the glands and other structures derivedtherefrom, e.g., corneal, esophegeal, epidermal, and hair follicleepithelial cells. Other exemplary epithlelial tissue includes: olfactoryepithelium, which is the pseudostratified epithelium lining theolfactory region of the nasal cavity, and containing the receptors forthe sense of smell; glandular epithelium, which refers to epitheliumcomposed of secreting cells; squamous epithelium, which refers toepithelium composed of flattened plate-like cells. The term epitheliumcan also refer to transitional epithelium, like that which ischaracteristically found lining hollow organs that are subject to greatmechanical change due to contraction and distention, e.g., tissue whichrepresents a transition between stratified squamous and columnarepithelium.

The term “epithelialization” refers to healing by the growth ofepithelial tissue over a denuded surface.

The term “epidermal gland” refers to an aggregation of cells associatedwith the epidermis and specialized to secrete or excrete materials notrelated to their ordinary metabolic needs. For example, “sebaceousglands” are holocrine glands in the corium that secrete an oilysubstance and sebum. The term “sweat glands” refers to glands thatsecrete sweat, situated in the corium or subcutaneous tissue, opening bya duct on the body surface.

The term “epidermis” refers to the outermost and nonvascular layer ofthe skin, derived from the embryonic ectoderm, varying in thickness from0.07-1.4 mm. On the palmar and plantar surfaces it comprises, fromwithin outward, five layers: basal layer composed of columnar cellsarranged perpendicularly; prickle-cell or spinous layer composed offlattened polyhedral cells with short processes or spines; granularlayer composed of flattened granular cells; clear layer composed ofseveral layers of clear, transparent cells in which the nuclei areindistinct or absent; and horny layer composed of flattened, cornifiednon-nucleated cells. In the epidermis of the general body surface, theclear layer is usually absent.

“Excisional wounds” include tears, abrasions, cuts, punctures orlacerations in the epithelial layer of the skin and may extend into thedermal layer and even into subcutaneous fat and beyond. Excisionalwounds can result from surgical procedures or from accidentalpenetration of the skin.

The “growth state” of a cell refers to the rate of proliferation of thecell and/or the state of differentiation of the cell. An “altered growthstate” is a growth state characterized by an abnormal rate ofproliferation, e.g., a cell exhibiting an increased or decreased rate ofproliferation relative to a normal cell.

The term “hair” refers to a threadlike structure, especially thespecialized epidermal structure composed of keratin and developing froma papilla sunk in the corium, produced only by mammals andcharacteristic of that group of animals. Also, “hair” may refer to theaggregate of such hairs. A “hair follicle” refers to one of thetubular-invaginations of the epidermis enclosing the hairs, and fromwhich the hairs grow. “Hair follicle epithelial cells” refers toepithelial cells that surround the dermal papilla in the hair follicle,e.g., stem cells, outer root sheath cells, matrix cells, and inner rootsheath cells. Such cells may be normal non-malignant cells, ortransformed/immortalized cells.

The term “hedgehog antagonist” refers to an agent that potentiates orrecapitulates the bioactivity of patched, such as to represstranscription of target genes. Preferred hedgehog antagonists can beused to overcome a ptc loss-of-function and/or a smoothenedgain-of-function, the latter also being referred to as smoothenedantagonists. The term ‘hedgehog antagonist’ as used herein refers notonly to any agent that may act by directly inhibiting the normalfunction of the hedgehog protein, but also to any agent that inhibitsthe hedgehog signalling pathway, and thus recapitulates the function ofptc.

The term “hedgehog gain-of-function” refers to an aberrant modificationor mutation of a ptc gene, hedgehog gene, or smoothened gene, or adecrease (or loss) in the level of expression of such a gene, whichresults in a phenotype which resembles contacting a cell with a hedgehogprotein, e.g., aberrant activation of a hedgehog pathway. Thegain-of-function may include a loss of the ability of the ptc geneproduct to regulate the level of expression of Ci genes, e.g., Gli1,Gli2, and Gli3. The term ‘hedgehog gain-of-function’ is also used hereinto refer to any similar cellular phenotype (e.g., exhibiting excessproliferation) that occurs due to an alteration anywhere in the hedgehogsignal transduction pathway, including, but not limited to, amodification or mutation of hedgehog itself. For example, a tumor cellwith an abnormally high proliferation rate due to activation of thehedgehog signalling pathway would have a ‘hedgehog gain-of-function’phenotype, even if hedgehog is not mutated in that cell.

As used herein, “immortalized cells” refers to cells that have beenaltered via chemical and/or recombinant means such that the cells havethe ability to grow through an indefinite number of divisions inculture.

“Internal epithelial tissue” refers to tissue inside the body that hascharacteristics similar to the epidermal layer in the skin. Examplesinclude the lining of the intestine. The method of the present inventionis useful for promoting the healing of certain internal wounds, forexample wounds resulting from surgery.

The term “keratosis” refers to proliferative skin disorder characterizedby hyperplasia of the horny layer of the epidermis. Exemplary keratoticdisorders include keratosis follicularis, keratosis palmaris etplantaris, keratosis pharyngea, keratosis pilaris, and actinickeratosis.

The term “LD₅₀” means the dose of a drug that is lethal in 50% of testsubjects.

The term “nail” refers to the horny cutaneous plate on the dorsalsurface of the distal end of a finger or toe.

The term “patched loss-of-function” refers to an aberrant modificationor mutation of a ptc gene, or a decreased level of expression of thegene, which results in a phenotype which resembles contacting a cellwith a hedgehog protein, e.g., aberrant activation of a hedgehogpathway. The loss-of-function may include a loss of the ability of theptc gene product to regulate the level of expression of Ci genes, e.g.,Gli1, Gli2 and Gli3. The term ‘ptc loss-of-function’ is also used hereinto refer to any similar cellular phenotype (e.g., exhibiting excessproliferation) that occurs due to an alteration anywhere in the hedgehogsignal transduction pathway, including, but not limited to, amodification or mutation of ptc itself. For example, a tumor cell withan abnormally high proliferation rate due to activation of the hedgehogsignalling pathway would have a ‘ptc loss-of-function’ phenotype, evenif ptc is not mutated in that cell.

A “patient” or “subject” to be treated by the subject method can meaneither a human or non-human animal.

The term “preventing” is art-recognized, and when used in relation to acondition, such as a local recurrence (e.g., pain), a disease such ascancer, a syndrome complex such as heart failure or any other medicalcondition, is well understood in the art, and includes administering,prior to onset of the condition, a composition that reduces thefrequency of, reduces the severity of, or delays the onset of symptomsof a medical condition in a subject relative to a subject which does notreceive the composition. Thus, prevention of cancer includes, forexample, reducing the number of detectable cancerous growths in apopulation of patients receiving a prophylactic treatment relative to anuntreated control population, and/or delaying the appearance ofdetectable cancerous growths in a treated population versus an untreatedcontrol population, e.g., by a statistically and/or clinicallysignificant amount. Prevention of an infection includes, for example,reducing the number of diagnoses of the infection in a treatedpopulation versus an untreated control population, and/or delaying theonset of symptoms of the infection in a treated population versus anuntreated control population. Prevention of pain includes, for example,reducing the frequency of, reducing the severity of, or alternativelydelaying, pain sensations experienced by subjects in a treatedpopulation versus an untreated control population.

The phrase “pharmaceutically acceptable salts” include both acid andbase addition salts. “Pharmaceutically acceptable acid addition salt”refers to those salts which retain the biological effectiveness andproperties of the free bases and which are not biologically or otherwiseundesirable, formed with inorganic acids such as hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoricacid and the like, and organic acids may be selected from aliphatic,cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic, andsulfonic classes of organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, gluconic acid, lactic acid, pyruvic acid,oxalic acid, malic acid, maleic acid, maloneic acid, succinic acid,fumaric acid, tartaric acid, citric acid, aspartic acid, ascorbic acid,glutamic acid, anthranilic acid, benzoic acid, cinnamic acid, mandelicacid, embonic acid, phenylacetic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicyclic acid and thelike.

The phrase “pharmaceutically acceptable base addition salts” includethose derived from inorganic bases such as sodium, potassium, lithium,ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminumsalts and the like. Particularly preferred are the ammonium, potassium,sodium, calcium and magnesium salts. Salts derived from pharmaceuticallyacceptable organic nontoxic bases includes salts of primary, secondary,and tertiary amines, substituted amines including naturally occurringsubstituted amines, cyclic amines and basic ion exchange resins, such asisopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine,dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,methylglucamine, theobromine, purines, piperizine, piperidine,N-ethylpiperidine, polyamine resins and the like. Particularly preferredorganic non-toxic bases are isopropylamine, diethylamine, ethanolamine,trimethamine, dicyclohexylamine, choline, and caffeine.

The term “prodrug” is intended to encompass compounds that, underphysiological conditions, are converted into the therapeutically activeagents of the present invention. A common method for making a prodrug isto include selected moieties that are hydrolyzed under physiologicalconditions to reveal the desired molecule. In other embodiments, theprodrug is converted by an enzymatic activity of the host animal.

As used herein, “proliferating” and “proliferation” refer to cellsundergoing mitosis.

Throughout this application, the term “proliferative skin disorder”refers to any disease/disorder of the skin marked by unwanted oraberrant proliferation of cutaneous tissue. These conditions aretypically characterized by epidermal cell proliferation or incompletecell differentiation, and include, for example, X-linked ichthyosis,psoriasis, atopic dermatitis, allergic contact dermatitis, epidermolytichyperkeratosis, and seborrheic dermatitis. For example,epidermodysplasia is a form of faulty development of the epidermis.Another example is “epidermolysis”, which refers to a loosened state ofthe epidermis with formation of blebs and bullae either spontaneously orat the site of trauma.

As used herein, the term “psoriasis” refers to a hyperproliferative skindisorder that alters the skin's regulatory mechanisms. In particular,lesions are formed which involve primary and secondary alterations inepidermal proliferation, inflammatory responses of the skin, and anexpression of regulatory molecules such as lymphokines and inflammatoryfactors. Psoriatic skin is morphologically characterized by an increasedturnover of epidermal cells, thickened epidermis, abnormalkeratinization, inflammatory cell infiltrates into the dermis layer andpolymorphonuclear leukocyte infiltration into the epidermis layerresulting in an increase in the basal cell cycle. Additionally,hyperkeratotic and parakeratotic cells are present.

The phrase “and salts and solvates thereof” as used herein means thatcompounds of the inventions may exist in one or a mixture of salts andsolvate forms. For example a compound of the invention may besubstantially pure in one particular salt or solvate form or else may bemixtures of two or more salt or solvate forms.

The term “skin” refers to the outer protective covering of the body,consisting of the corium and the epidermis, and is understood to includesweat and sebaceous glands, as well as hair follicle structures.Throughout the present application, the adjective “cutaneous” may beused, and should be understood to refer generally to attributes of theskin, as appropriate to the context in which they are used.

The term “smoothened gain-of-function” refers to an aberrantmodification or mutation of a smo gene, or an increased level ofexpression of the gene, which results in a phenotype that resemblescontacting a cell with a hedgehog protein, e.g., aberrant activation ofa hedgehog pathway. While not wishing to be bound by any particulartheory, it is noted that ptc may not signal directly into the cell, butrather interact with smoothened, another membrane-bound protein locateddownstream of ptc in hedgehog signaling (Marigo et al., (1996) Nature384: 177-179). The gene smo is a segment-polarity gene required for thecorrect patterning of every segment in Drosophila (Alcedo et al., (1996)Cell 86: 221-232). Human homologs of smo have been identified. See, forexample, Stone et al. (1996) Nature 384:129-134, and GenBank accessionU84401. The smoothened gene encodes an integral membrane protein withcharacteristics of heterotrimeric G-protein-coupled receptors; i.e.,7-transmembrane regions. This protein shows homology to the DrosophilaFrizzled (Fz) protein, a member of the wingless pathway. It wasoriginally thought that smo encodes a receptor of the Hh signal.However, this suggestion was subsequently disproved, as evidence for ptcbeing the Hh receptor was obtained. Cells that express Smo fail to bindHh, indicating that smo does not interact directly with Hh (Nusse,(1996) Nature 384: 119-120). Rather, the binding of Sonic hedgehog (SHH)to its receptor, PTCH, is thought to prevent normal inhibition by PTCHof smoothened (SMO), a seven-span transmembrane protein.

Recently, it has been reported that activating smoothened mutationsoccur in sporadic basal cell carcinoma, Xie et al. (1998) Nature 391:90-2, and primitive neuroectodermal tumors of the central nervoussystem, Reifenberger et al. (1998) Cancer Res 58: 1798-803.

The term “therapeutic index” refers to the therapeutic index of a drugdefined as LD₅₀/ED₅₀.

As used herein, “transformed cells” refers to cells that havespontaneously converted to a state of unrestrained growth, i.e., theyhave acquired the ability to grow through an indefinite number ofdivisions in culture. Transformed cells may be characterized by suchterms as neoplastic, anaplastic and/or hyperplastic, with respect totheir loss of growth control.

The term “treating” as used herein includes prophylaxis of the namedcondition or amelioration or elimination of the condition once it hasbeen established.

The term “acyl” means a carbonyl containing substituent represented bythe formula —C(O)—R in which R is alkyl, a carbocycle, a heterocycle,carbocycle-substituted alkyl or heterocycle-substituted alkyl whereinalkyl, carbocycle and heterocycle are as herein defined. Acyl groupsinclude alkanoyl (e.g. acetyl), aroyl (e.g. benzoyl), and heteroaroyl.

The term “acylamino” is art-recognized and refers to a moiety that canbe represented by the general formula:

wherein R₉ is as defined above, and R′₁₁ represents a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)—R₈, where m and R₈ are as defined above.

The term “acyloxy” is art-recognized and refers to a moiety that can berepresented by the general formula:

wherein R′₁₁ represents a hydrogen, an alkyl, an alkenyl or—(CH₂)_(m)—R₈, where m and R₈ are as defined above.

Herein, the term “aliphatic group” refers to a straight-chain,branched-chain, or cyclic aliphatic hydrocarbon group and includessaturated and unsaturated aliphatic groups, such as an alkyl group, analkenyl group, and an alkynyl group.

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl, —O—(CH₂)_(m)—R₈,where m and R₈ are described above.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, andcycloalkyl-substituted alkyl groups. In preferred embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C₁-C₃₀ for straight chains, C₃-C₃₀ for branchedchains), and more preferably 20 or fewer. Likewise, preferredcycloalkyls have from 3-10 carbon atoms in their ring structure, andmore preferably have 5, 6 or 7 carbons in the ring structure.

Moreover, the term “alkyl” (or “lower alkyl”) as used throughout thespecification, examples, and claims is intended to include both“unsubstituted alkyls” and “substituted alkyls”, the latter of whichrefers to alkyl moieties having substituents replacing a hydrogen on oneor more carbons of the hydrocarbon backbone. Such substituents caninclude, for example, a halogen, a hydroxyl, a carbonyl (such as acarboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (suchas a thioester, a thioacetate, or a thioformate), an alkoxyl, aphosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, anamido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl,an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, asulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromaticmoiety. It will be understood by those skilled in the art that themoieties substituted on the hydrocarbon chain can themselves besubstituted, if appropriate. For instance, the substituents of asubstituted alkyl may include substituted and unsubstituted forms ofamino, azido, imino, amido, phosphoryl (including phosphonate andphosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl andsulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls(including ketones, aldehydes, carboxylates, and esters), —CF₃, —CN andthe like. Exemplary substituted alkyls are described below. Cycloalkylscan be further substituted with alkyls, alkenyls, alkoxys, alkylthios,aminoalkyls, carbonyl-substituted alkyls, —CF₃, —CN, and the like.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths. Throughout the application, preferred alkylgroups are lower alkyls. In preferred embodiments, a substituentdesignated herein as alkyl is a lower alkyl.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In preferred embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl,—S-alkynyl, and —S—(CH₂)_(m)—R₈, wherein m and R₈ are defined above.Representative alkylthio groups include methylthio, ethylthio, and thelike.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that can berepresented by the general formula:

wherein R₉, R₁₀ and R′₁₀ each independently represent a hydrogen, analkyl, an alkenyl, —(CH₂)_(m)—R₈, or R₉ and R₁₀ taken together with theN atom to which they are attached complete a heterocycle having from 4to 8 atoms in the ring structure; R₈ represents an aryl, a cycloalkyl, acycloalkenyl, a heterocycle or a polycycle; and m is zero or an integerin the range of 1 to 8. In preferred embodiments, only one of R₉ or R₁₀can be a carbonyl, e.g., R₉, R₁₀ and the nitrogen together do not forman imide. In certain such embodiments, neither R₉ and R₁₀ is attached toN by a carbonyl, e.g., the amine is not an amide or imide, and the amineis preferably basic, e.g., its conjugate acid has a pK_(a) above 7. Ineven more preferred embodiments, R₉ and R₁₀ (and optionally R′₁₀) eachindependently represent a hydrogen, an alkyl, an alkenyl, or—(CH₂)_(m)—R₈. Thus, the term “alkylamine” as used herein means an aminegroup, as defined above, having a substituted or unsubstituted alkylattached thereto, i.e., at least one of R₉ and R₁₀ is an alkyl group.

The term “amino-protecting group” as Used herein refers to a derivativeof the groups commonly employed to block or protect an amino group whilereactions are carried out on other functional groups on the compound.Examples of such protecting groups include carbamates, amides, alkyl andaryl groups, imines, as well as many N-heteroatom derivatives which canbe removed to regenerate the desired amine group. Preferred aminoprotecting groups are Boc, Fmoc and Cbz. Further examples of thesegroups are found in T. W. Greene and P. G. M. Wuts, “Protective Groupsin Organic Synthesis”, 2^(nd) ed., John Wiley & Sons, Inc., New York,N.Y., 1991, chapter 7; E. Haslam, “Protective Groups in OrganicChemistry”, J. G. W. McOmie, Ed., Plenum Press, New York, N.Y., 1973,Chapter 5, and T. W. Greene, “Protective Groups in Organic Synthesis”,John Wiley and Sons, New York, N.Y., 1981. The term “protected amino”refers to an amino group substituted with one of the aboveamino-protecting groups.

The term “amido” is art-recognized as an amino-substituted carbonyl andincludes a moiety that can be represented by the general formula:

wherein R₉, R₁₀ are as defined above. Preferred embodiments of the amidewill not include imides that may be unstable.

The term “amidine” denotes the group —C(NH)—NHR wherein R is H or alkylor aralkyl. A preferred amidine is the group —C(NH)—NH₂. The term“aralkyl”, as used herein, refers to an alkyl group substituted with anaryl group (e.g., an aromatic or heteroaromatic group).

The term “aryl” as used herein includes 5-, 6-, and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “aryl heterocycles” or“heteroaromatics.” The aromatic ring can be substituted at one or morering positions with such substituents as described above, for example,halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF₃, —CN, or the like. The term “aryl” alsoincludes polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings (the ringsare “fused rings”) wherein at least one of the rings is aromatic, e.g.,the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls and/or heterocyclyls.

The term “carbocycle”, as used herein, refers to an aromatic ornon-aromatic ring in which each atom of the ring is carbon.

The term “carboxy-protecting group” as used herein refers to one of theester derivatives of the carboxylic acid group commonly employed toblock or protect the carboxylic acid group while reactions are carriedout on other functional groups on the compound. Examples of suchcarboxylic acid protecting groups include 4-nitrobenzyl,4-methoxybenzyl, 3,4-dimethoxybenzyl, 2,4-dimethoxybenzyl,2,4,6-trimethoxybenzyl, 2,4,6-trimethylbenzyl, pentamethylbenzyl,3,4-methylenedioxybenzyl, benzhydryl, 4,4′-dimethoxybenzhydryl,2,2′,4,4′-tetramethoxybenzhydryl, alkyl such as t-butyl or t-amyl,trityl, 4-methoxytrityl, 4,4′-dimethoxytrityl, 4,4′,4″-trimethoxytrityl,2-phenylprop-2-yl, trimethylsilyl, t-butyldimethylsilyl, phenacyl,2,2,2-trichloroethyl, beta-(trimethylsilyl)ethyl,beta-(di(n-butyl)methylsilyl)ethyl, p-toluenesulfonylethyl,4-nitrobenzylsulfonylethyl, allyl, cinnamyl,1-(trimethylsilylmethyl)prop-1-en-3-yl, and like moieties. The speciesof carboxy-protecting group employed is not critical so long as thederivatized carboxylic acid is stable to the condition of subsequentreaction(s) on other positions of the molecule and can be removed at theappropriate point without disrupting the remainder of the molecule. Inparticular, it is important not to subject a carboxy-protected moleculeto strong nucleophilic bases, such as lithium hydroxide or NaOH, orreductive conditions employing highly activated metal hydrides such asLiAlH₄. (Such harsh removal conditions are also to be avoided whenremoving amino-protecting groups and hydroxy-protecting groups,discussed below.) Preferred carboxylic acid protecting groups are thealkyl (e.g. methyl, ethyl, t-butyl), allyl, benzyl and p-nitrobenzylgroups. Similar carboxy-protecting groups used in the cephalosporin,penicillin and peptide arts can also be used to protect a carboxy groupsubstituents. Further examples of these groups are found in T. W. Greeneand P. G. M. Wuts, “Protective Groups in Organic Synthesis”, 2^(nd) ed.,John Wiley & Sons, Inc., New York, N.Y., 1991, chapter 5; E. Haslam,“Protective Groups in Organic Chemistry”, J. G. W. McOmie, Ed., PlenumPress, New York, N.Y., 1973, Chapter 5, and T. W. Greene, “ProtectiveGroups in Organic Synthesis”, John Wiley and Sons, New York, N.Y., 1981,Chapter 5. The term “protected carboxy” refers to a carboxy groupsubstituted with one of the above carboxy-protecting groups.

The term “carbonyl” is art-recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R₁₁represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R₈ or apharmaceutically acceptable salt, R′₁₁ represents a hydrogen, an alkyl,an alkenyl or —(CH₂)_(m)—R₈, where m and R₈ are as defined above. WhereX is an oxygen and R₁₁ or R′₁₁ is not hydrogen, the formula representsan “ester”. Where X is an oxygen, and R₁₁ is as defined above, themoiety is referred to herein as a carboxyl group, and particularly whenR₁₁ is a hydrogen, the formula represents a “carboxylic acid”. Where Xis an oxygen, and R′₁₁ is hydrogen, the formula represents a “formate”.In general, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiocarbonyl” group. Where X is asulfur and R₁₁ or R′₁₁ is not hydrogen, the formula represents a“thioester.” Where X is a sulfur and R₁₁ is hydrogen, the formularepresents a “thiocarboxylic acid.” Where X is a sulfur and R₁₁′ ishydrogen, the formula represents a “thiolformate.” On the other hand,where X is a bond, and R₁₁ is not hydrogen, the above formula representsa “ketone” group.

Where X is a bond, and R₁₁ is hydrogen, the above formula represents an“aldehyde” group.

The term guanidine” denotes the group —NH—C(NH)—NHR wherein R is H oralkyl or aralkyl. A particular guanidine group is —NH—C(NH)—NH₂.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are boron, nitrogen,oxygen, phosphorus, sulfur and selenium.

The terms “heterocycle,” “heterocyclyl” or “heterocyclic group” refer to3- to 10-membered ring structures, more preferably 3- to 7-memberedwings, whose ring structures include one to four heteroatoms.Heterocycles can also be polycycles.

Heterocyclyl groups include, for example, thiophene, thianthrene, furan,pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole,imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine,quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine,quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,phenanthridine, acridine, pyrimidine, phenanthroline, phenazine,phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane,thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactamssuch as azetidinones and pyrrolidinones, sultams, sultones, and thelike. The heterocyclic ring can be substituted at one or more positionswith such substituents as described above, as for example, halogen,alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl,carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, aheterocyclyl, an aromatic or heteroaromatic moiety, —CF₃, —CN, or thelike.

The term “hydroxy-protecting group” as used herein refers to aderivative of the hydroxy group commonly employed to block or protectthe hydroxy group while reactions are carried out on other functionalgroups on the compound. Examples of such protecting groups includetetrahydropyranyloxy, benzoyl, acetoxy, carbamoyloxy, benzyl, andsilylethers (e.g. TBS, TBDPS) groups. Further examples of these groupsare found in T. W. Greene and P. G. M. Wuts, “Protective Groups inOrganic Synthesis”, 2^(nd) ed., John Wiley & Sons, Inc., New York, N.Y.,1991, chapters 2-3; E. Haslam, “Protective Groups in Organic Chemistry”,J. G. W. McOmie, Ed., Plenum Press, New York, N.Y., 1973, Chapter 5, andT. W. Greene, “Protective Groups in Organic Synthesis”, John Wiley andSons, New York, N.Y., 1981. The term “protected hydroxy” refers to ahydroxy group substituted with one of the above hydroxy-protectinggroups.

As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term“hydroxyl” means —OH; and the term “sulfonyl” means —SO₂—.

A “phosphonamidite” can be represented in the general formula:

wherein R₉ and R₁₀ are as defined above, Q₂ represents O, S or N, andR₄₈ represents a lower alkyl or an aryl, Q₂ represents O, S or N.

A “phosphoramidite” can be represented in the general formula:

wherein R₉ and R₁₀ are as defined above, and Q₂ represents O, S or N.

A “phosphoryl” can in general be represented by the formula:

wherein Q₁ represented S or O, and R₄₆ represents hydrogen, a loweralkyl or an aryl. When used to substitute, for example, an alkyl, thephosphoryl group of the phosphorylalkyl can be represented by thegeneral formula:

wherein Q₁ represented S or O, and each R₄₆ independently representshydrogen, a lower alkyl or an aryl, Q₂ represents O, S or N. When Q₁ isan S, the phosphoryl moiety is a “phosphorothioate”.

The terms “polycyclyl” or “polycyclic group” refer to two or more rings(e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Rings that are joined throughnon-adjacent atoms are termed “bridged” rings. Each of the rings of thepolycycle can be substituted with such substituents as described above,as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic orheteroaromatic moiety, —CF₃, —CN, or the like.

The phrase “protecting group” as used herein means temporarysubstituents which protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G.M. Protective Groups in Organic Synthesis, 2^(nd) ed.; Wiley: New York,1991).

A “selenoalkyl” refers to an alkyl group having a substituted selenogroup attached thereto. Exemplary “selenoethers” which may besubstituted on the alkyl are selected from one of —Se-alkyl,—Se-alkenyl, —Se-alkynyl, and —Se—(CH₂)_(m)—R₈, m and R₈ being definedabove.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described herein above. The permissible substituentscan be one or more and the same or different for appropriate organiccompounds. For purposes of this invention, the heteroatoms such asnitrogen may have hydrogen substituents and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalences of the heteroatoms. This invention is not intended to belimited in any manner by the permissible substituents of organiccompounds.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

The term “sulfamoyl” is art-recognized and includes a moiety that can berepresented by the general formula:

in which R₉ and R₁₀ are as defined above.

The term “sulfate” is art recognized and includes a moiety that can berepresented by the general formula:

in which R₄₁ is as defined above.

The term “sulfonamido” is art recognized and includes a moiety that canbe represented by the general formula:

in which R₉ and R′₁₁ are as defined above.

The term “sulfonate” is art-recognized and includes a moiety that can berepresented by the general formula:

in which R₄₁ is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The terms “sulfoxido” or “sulfinyl”, as used herein, refers to a moietythat can be represented by the general formula:

in which R₄₄ is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.

Analogous substitutions can be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

As used herein, the definition of each expression, e.g., alkyl, m, n,etc., when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized andrefer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl,and nonafluorobutanesulfonyl groups, respectively. The terms triflate,tosylate, mesylate, and nonaflate are art-recognized and refer totrifluoromethanesulfonate ester, p-toluenesulfonate ester,methanesulfonate ester, and nonafluorobutanesulfonate ester functionalgroups and molecules that contain said groups, respectively.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl,phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations. The abbreviationscontained in said list, and all abbreviations utilized by organicchemists of ordinary skill in the art are hereby incorporated byreference.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts may be formed with an appropriateoptically active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

Contemplated equivalents of the compounds described above includecompounds which otherwise correspond thereto, and which have the samegeneral properties thereof (e.g., the ability to inhibit hedgehogsignaling), wherein one or more simple variations of substituents aremade which do not adversely affect the efficacy of the compound. Ingeneral, the compounds of the present invention may be prepared by themethods illustrated in the general reaction schemes as, for example,described below, or by modifications thereof, using readily availablestarting materials, reagents and conventional synthesis procedures. Inthese reactions, it is also possible to make use of variants which arein themselves known, but are not mentioned here.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover. Alsofor purposes of this invention, the term “hydrocarbon” is contemplatedto include all permissible compounds having at least one hydrogen andone carbon atom. In a broad aspect, the permissible hydrocarbons includeacyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic organic compounds which can besubstituted or unsubstituted.

III. Exemplary Compounds of the Invention

As described in further detail below, it is contemplated that thesubject methods can be carried out using a variety of different smallmolecules which can be readily identified, for example, by such drugscreening assays as described herein. For example, compounds useful inthe subject methods include compounds may be represented by generalformula (I):

wherein, as valence and stability permit,

X and Z, independently, represent —N(R₇)—, —O—, —S—, —(R₇)N—N(R₇)—,—ON(R₇)—, or a direct bond, preferably —N(R₇)—, —O—, —S—, or a directbond;

Y represents —C(═O)—, —C(═S)—, —C(═NR₇)—, SO₂, or SO, preferably—C(═O)—, SO₂, or —C(═S)—;

A represents O, S, or NR₇, preferably O or NH, and most preferably NH;

G represents a cycloalkyl, heterocyclyl, aryl, or heteroaryl ring fusedto the ring to which it is attached, preferably an aryl or heteroarylring.

Ar represents a substituted or unsubstituted aryl or heteroaryl ring,such as a substituted or unsubstituted phenyl ring;

R₁, represents a substituted or unsubstituted pyridine ring;

R₂ represents from 0-4 substituents on the ring to which it is attached,such as halogen, lower alkyl, lower alkenyl, aryl, heteroaryl, carbonylgroup (e.g., ester, carboxyl, or formyl), thiocarbonyl (e.g., thioester,thiocarboxylate, or thioformate), ketone, aldehyde, amino, acylamino,amido, amidino, cyano, nitro, azido, sulfonyl, sulfoxido, sulfate,sulfonate, sulfamoyl, sulfonamido, phosphoryl, phosphonate, phosphinate,J-R₈, J-OH, J-lower alkyl, J-lower alkenyl, J-R₈, J-SH, J-NH₂, protectedforms of the above, or any two R₂, when occurring more than once in acyclic or polycyclic structure, can be taken together form a 4- to8-membered cycloalkyl, aryl, or heteroaryl;

R₇, independently for each occurrence, represents H, lower alkyl (e.g.,substituted or unsubstituted), J-cycloalkyl (e.g., substituted orunsubstituted), J-heterocyclyl (e.g., substituted or unsubstituted),J-aryl (e.g., substituted or unsubstituted), J-heteroaryl (e.g.,substituted or unsubstituted);

R₈, independently for each occurrence, represents H, lower alkyl (e.g.,substituted or unsubstituted), cycloalkyl (e.g., substituted orunsubstituted), heterocyclyl (e.g., substituted or unsubstituted), aryl(e.g., substituted or unsubstituted), or heteroaryl (e.g., substitutedor unsubstituted); and

J represents, independently for each occurrence, a chain having from 0-8(preferably from 0-4) units selected from CK₂, NK, O, and S, wherein Krepresents, independently for each occurrence, H or lower alkyl.

In certain embodiments, at least one of Z and X is not a direct bond. Incertain embodiments, X-Y-Z includes an amide, urea, or sulfonamide. Incertain embodiments, X is selected from —N(R₈)—, —O—, —S—, andpreferably represents NH.

In certain embodiments, R₁ is a pyridine ring, optionally substitutedwith from 1-5 substituents, such as nitro, halogen, cyano, lower alkyl(such as methyl or halogenated lower alkyl (such as trifluoromethyl)),alkenyl, alkynyl, aralkyl, amino, alkylamino, acylamino (e.g.,R₈—C(═O)NH—), amido, hydroxyl, alkoxy, acyloxy (e.g., R₈—C(═O)—O—),carbonyl, phosphoryl, sulfamoyl, sulfate, sulfonamide, sulfonate,sulfoxido, sulfhydryl, sulfonyl, a substituted or unsubstitutedcycloalkyl, heterocyclyl, aryl, or heteroaryl fused to the pyridinering. In certain embodiments, R₁ is a pyridine ring, optionallysubstituted with from 1-5 substituents, such as nitro, cyano, loweralkyl (such as methyl or halogenated lower alkyl (such astrifluoromethyl)), alkenyl, alkynyl, aralkyl, amino, alkylamino,acylamino (e.g., R₈—C(═O)NH—), amido, hydroxyl, alkoxy, acyloxy (e.g.,R₈—C(═O)—O—), carbonyl, phosphoryl, sulfamoyl, sulfate, sulfonamide,sulfonate, sulfoxido, sulfhydryl, or sulfonyl. In certain embodiments,one or more of the substituents on the pyridine ring is bound to aprotecting group. In some embodiments, the pyridine ring is substitutedwith a methyl, trifluoromethyl, or both.

The pyridine ring of R¹ can be substituted at any position on the ring,for example ortho, meta or para to the nitrogen atom of the pyridinering. In some embodiments, the pyridine ring is substituted with twosubstituents in addition to J (disubstituted). These two substituentscan have any positional relationship with respect to one another allowedby the pyridine ring. For example, these substituents can be in a orthorelationship to each other (e.g., 2,3; 3,4; 4,5; or 5,6). They can havea meta relationship to each other (e.g., 2,4; 2,6; 3,5; or 4,6). Or theycan have a para relationship (e.g., 2,5; or 3,6). In other embodiments,the pyridine ring is substituted with three substituents in addition toJ (trisubstituted). These substituents also can have any positionalrelationship respect to one another allowed by the pyridine ring. In yetother embodiments, the pyridine ring is substituted with foursubstituents in addition to J (tetrasubstituted). These substituentsalso can have any positional relationship respect to one another allowedby the pyridine ring.

In certain embodiments the pyridine ring of R₁ can be substituted on aposition orthro to the nitrogen atom. In further embodiments, thepyridine ring can be substituted at both positions ortho to the nitrogenatom. The pyridine ring can have either position ortho to the nitrogenatom substituted by a methyl or trifluoromethyl. In another embodiment,one position ortho to the nitrogen of the pyridine ring is substitutedwith methyl and the other position ortho to the nitrogen is substitutedwith trifluoromethyl.

The pyridine ring of R₁ can be connected to J at any position on thering. For example, the pyridine ring can be connected to J ortho to thenitrogen atom of the pyridine ring (the C2 position, i.e., a 2-pyridylring), meta to the nitrogen atom of the pyridine ring (the C3 position,i.e., a 3-pyridyl ring), or para to the nitrogen atom of the pyridinering (the C4 position, i.e., a 4-pyridyl ring). The different positionsfor connection to J can be contemplated with the diverse substitutionpossibilities disclosed above. For example, in one embodiment, thepyridine ring is connected to J meta to the nitrogen atom and the ringis substituted with two additional substituents (disubstituted), such asmethyl and trifluoromethyl.

The nitrogen atom of the pyridine ring of R₁ may also be substitutedwith heteroatoms, such as oxygen (e.g., producing a nitrone or N-oxide).Substitution of the nitrogen atom of the pyridine ring may be incombination with additional substitution on the pyridine ring.

In certain embodiments, X and the ring comprising A are disposed on Arin a meta (i.e., 1,3) relationship.

In certain embodiments, G represents a phenyl or piperidine ring.

In certain embodiments, J is absent.

In certain embodiments, R₂ represents from 1-4 substituents selectedfrom halogen, cyano, nitro, alkoxy, amino, acylamino (e.g.,R₈—C(═O)NH—), a substituted or unsubstituted cycloalkyl, heterocyclyl,aryl, or heteroaryl fused to G, and substituted or unsubstituted loweralkyl.

In certain embodiments, compounds useful in the present invention may berepresented by general formula (II):

wherein, as valence and stability permit,

X and Z, independently, represent —N(R₇)—, —O—, —S—, —(R₇)N—N(R₇)—,—ON(R₇)— or a direct bond, preferably —N(R₇)—, —O—, —S—, or a directbond;

Y represents —C(═O)—, —C(═S)—, —C(═NR₇)—, SO₂, or SO, preferably—C(═O)—, SO₂, or —C(═S)—;

A represents O, S, or NR₇, preferably O or NH, and most preferably NH;

G represents a cycloalkyl, heterocyclyl, aryl, or heteroaryl ring fusedto the ring to which it is attached, preferably an aryl or heteroarylring.

R₁ represents a substituted or unsubstituted pyridine ring;

R₂ represents from 0-4 substituents on the ring to which it is attached,such as halogen, lower alkyl, lower alkenyl, aryl, heteroaryl, carbonylgroup (e.g., ester, carboxyl, or formyl), thiocarbonyl (e.g., thioester,thiocarboxylate, or thioformate), ketone, aldehyde, amino, acylamino,amido, amidino, cyano, nitro, azido, sulfonyl, sulfoxido, sulfate,sulfonate, sulfamoyl, sulfonamido, phosphoryl, phosphonate, phosphinate,J-R₈, J-OH, J-lower alkyl, J-lower alkenyl, J-R₈, J-SH, J-NH₂, protectedforms of the above, or any two R₂, when occurring more than once in acyclic or polycyclic structure, can be taken together form a 4- to8-membered cycloalkyl, aryl, or heteroaryl;

R₃ represents from 0-4 substituents on the ring to which it is attached,such as halogen, hydroxyl, alkoxy, amino, alkylamino, cyano, nitro,substituted or unsubstituted lower alkyl, and acyl, preferably halogen,lower alkoxy, or substituted or unsubstituted lower alkyl;

R₇, independently for each occurrence, represents H, lower alkyl (e.g.,substituted or unsubstituted), J-cycloalkyl (e.g., substituted orunsubstituted), J-heterocyclyl (e.g., substituted or unsubstituted),J-aryl (e.g., substituted or unsubstituted), J-heteroaryl (e.g.,substituted or unsubstituted);

R₈, independently for each occurrence, represents H, lower alkyl (e.g.,substituted or unsubstituted), cycloalkyl (e.g., substituted orunsubstituted), heterocyclyl (e.g., substituted or unsubstituted), aryl(e.g., substituted or unsubstituted), or heteroaryl (e.g., substitutedor unsubstituted); and

J represents, independently for each occurrence, a chain having from 0-8(preferably from 0-4) units selected from CK₂, NK, O, and S, wherein Krepresents, independently for each occurrence, H or lower alkyl.

In certain embodiments, at least one of Z and X is not a direct bond. Incertain embodiments, X-Y-Z includes an amide, urea, or sulfonamide. Incertain embodiments, X is selected from —N(R₈)—, —O—, —S—, andpreferably represents NH.

In certain embodiments, R₁ includes a pyridine ring, optionallysubstituted with from 1-5 substituents, such as nitro, halogen, cyano,lower alkyl, acylamino (e.g., R₈—C(═O)NH—), alkoxy, alkylamino, asubstituted or unsubstituted cycloalkyl, heterocyclyl, aryl, orheteroaryl fused to the pyridine ring. In some embodiments, the pyridinering is connected to J at the C3 position (i.e., a 3-pyridyl ring). Insome embodiments, the pyridine ring may be substituted at the C2position, the C6 position, or both. In some embodiments, the pyridinering is substituted with a methyl group or a trifluoromethyl group orboth.

In certain embodiments, G represents a phenyl or piperidine ring.

In certain embodiments, J is absent.

In certain embodiments, R₂ represents from 1-4 substituents selectedfrom halogen, cyano, nitro, alkoxy, amino, acylamino (e.g.,R₈—C(═O)NH—), a substituted or unsubstituted cycloalkyl, heterocyclyl,aryl, or heteroaryl fused to G, and substituted or unsubstituted loweralkyl.

In certain embodiments, R₃ includes a substituent, such as a substitutedor unsubstituted alkyl or a halogen, at a position para either to X orto the ring including A.

As described in further detail below, it is contemplated that thesubject methods can be carried out using a variety of different smallmolecules which can be readily identified, for example, by such drugscreening assays as described herein. For example, compounds useful inthe subject methods include compounds may be represented by generalformula (III):

wherein, as valence and stability permit,

X and Z, independently, represent —N(R₇)—, —O—, —S—, —(R₇)N—N(R₇)—,—ON(R₇)—, or a direct bond, preferably —N(R₇)—, —O—, —S—, or a directbond;

Y represents —C(═O)—, —C(═S)—, —C(═NR₇)—, SO₂, or SO, preferably—C(═O)—, SO₂, or —C(═S)—;

A represents O, S, or NR₇, preferably O or NH, and most preferably NH;

G represents a cycloalkyl, heterocyclyl, aryl, or heteroaryl ring fusedto the ring to which it is attached, preferably an aryl or heteroarylring;

Q is absent, or represents CK₂, NK, O, and S, wherein K represents,independently for each occurrence, H or lower alkyl;

Ar represents a substituted or unsubstituted aryl or heteroaryl ring,such as a substituted or unsubstituted phenyl ring;

R₁, represents a substituted or unsubstituted pyridine ring;

R₂ represents from 0-4 substituents on the ring to which it is attached,such as halogen, lower alkyl, lower alkenyl, aryl, heteroaryl, carbonylgroup (e.g., ester, carboxyl, or formyl), thiocarbonyl (e.g., thioester,thiocarboxylate, or thioformate), ketone, aldehyde, amino, acylamino,amido, amidino, cyano, nitro, azido, sulfonyl, sulfoxido, sulfate,sulfonate, sulfamoyl, sulfonamido, phosphoryl, phosphonate, phosphinate,J-R₈, J-OH, J-lower alkyl, J-lower alkenyl, J-R₈, J-SH, J-NH₂, protectedforms of the above, or any two R₂, when occurring more than once in acyclic or polycyclic structure, can be taken together form a 4- to8-membered cycloalkyl, aryl, or heteroaryl;

R₇, independently for each occurrence, represents H, lower alkyl (e.g.,substituted or unsubstituted), J-cycloalkyl (e.g., substituted orunsubstituted), J-heterocyclyl (e.g., substituted or unsubstituted),J-aryl (e.g., substituted or unsubstituted), J-heteroaryl (e.g.,substituted or unsubstituted);

R₈, independently for each occurrence, represents H, lower alkyl (e.g.,substituted or unsubstituted), cycloalkyl (e.g., substituted orunsubstituted), heterocyclyl (e.g., substituted or unsubstituted), aryl(e.g., substituted or unsubstituted), or heteroaryl (e.g., substitutedor unsubstituted); and

J represents, independently for each occurrence, a chain having from 0-8(preferably from 0-4) units selected from CK₂, NK, O, and S.

In certain embodiments, at least one of Z and X is not a direct bond. Incertain embodiments, X-Y-Z includes an amide, urea, or sulfonamide. Incertain embodiments, X is selected from —N(R₈)—, —O—, —S—, andpreferably represents NH.

In certain embodiments, R₁, includes a pyridine ring, optionallysubstituted with from 1-5 substituents, such as nitro, halogen, cyano,lower alkyl, acylamino (e.g., R₈—C(═O)NH—), alkoxy, alkylamino, asubstituted or unsubstituted cycloalkyl, heterocyclyl, aryl, orheteroaryl fused to the pyridine ring. In some embodiments, the pyridinering is connected to J at the C3 position (i.e., a 3-pyridyl ring). Insome embodiments, the pyridine ring may be further substituted at the C2position or at the C6 position or both. In some embodiments the pyridinering is substituted with a methyl group or a trifluoromethyl group orboth.

In certain embodiments, X and the ring comprising A are disposed on Arin a meta (i.e., 1,3) relationship.

In certain embodiments, G represents a phenyl or piperidine ring.

In certain embodiments, J is absent.

In certain embodiments, R₂ represents from 1-4 substituents selectedfrom halogen, cyano, nitro, alkoxy, amino, acylamino (e.g.,R₈—C(═O)NH—), a substituted or unsubstituted cycloalkyl, heterocyclyl,aryl, or heteroaryl fused to G, and substituted or unsubstituted loweralkyl.

In certain embodiments, compounds useful in the present invention may berepresented by general formula (IV):

wherein, as valence and stability permit,

X and Z, independently, represent —N(R₇)—, —O—, —S—, —(R₇)N—N(R₇)—,—ON(R₇)— or a direct bond, preferably —N(R₇)—, —O—, —S—, or a directbond;

Y represents —C(═O)—, —C(═S)—, —C(—NR₇)—, SO₂, or SO, preferably—C(═O)—, SO₂, or —C(═S)—;

A represents O, S, or NR₇, preferably O or NH, and most preferably NH;

G represents a cycloalkyl, heterocyclyl, aryl, or heteroaryl ring fusedto the ring to which it is attached, preferably an aryl or heteroarylring.

Q is absent, or represents CK₂, NK, O, and S, wherein K represents,independently for each occurrence, H or lower alkyl;

R₁ represents a substituted or unsubstituted pyridine ring;

R₂ represents from 0-4 substituents on the ring to which it is attached,such as halogen, lower alkyl, lower alkenyl, aryl, heteroaryl, carbonylgroup (e.g., ester, carboxyl, or formyl), thiocarbonyl (e.g., thioester,thiocarboxylate, or thioformate), ketone, aldehyde, amino, acylamino,amido, amidino, cyano, nitro, azido, sulfonyl, sulfoxido, sulfate,sulfonate, sulfamoyl, sulfonamido, phosphoryl, phosphonate, phosphinate,J-R₈, J-OH, J-lower alkyl, J-lower alkenyl, J-R₈, J-SH, J-NH₂, protectedforms of the above, or any two R₂, when occurring more than once in acyclic or polycyclic structure, can be taken together form a 4- to8-membered cycloalkyl, aryl, or heteroaryl;

R₃ represents from 0-4 substituents on the ring to which it is attached,such as halogen, hydroxyl, alkoxy, amino, alkylamino, cyano, nitro,substituted or unsubstituted lower alkyl, and acyl, preferably halogen,lower alkoxy, or substituted or unsubstituted lower alkyl;

R₇, independently for each occurrence, represents H, lower alkyl (e.g.,substituted or unsubstituted), J-cycloalkyl (e.g., substituted orunsubstituted), J-heterocyclyl (e.g., substituted or unsubstituted),J-aryl (e.g., substituted or unsubstituted), J-heteroaryl (e.g.,substituted or unsubstituted);

R₈, independently for each occurrence, represents H, lower alkyl (e.g.,substituted or unsubstituted), cycloalkyl (e.g., substituted orunsubstituted), heterocyclyl (e.g., substituted or unsubstituted), aryl(e.g., substituted or unsubstituted), or heteroaryl (e.g., substitutedor unsubstituted); and

J represents, independently for each occurrence, a chain having from 0-8(preferably from 0-4) units selected from CK₂, NK, O, and S.

In certain embodiments, at least one of Z and X is not a direct bond. Incertain embodiments, X-Y-Z includes an amide, urea, or sulfonamide. Incertain embodiments, X is selected from —N(R₈)—, —O—, —S—, andpreferably represents NH.

In certain embodiments, R₁ includes a pyridine ring, optionallysubstituted with from 1-5 substituents, such as nitro, halogen, cyano,lower alkyl, acylamino (e.g., R₈—C(═O)NH—), alkoxy, alkylamino, asubstituted or unsubstituted cycloalkyl, heterocyclyl, aryl, orheteroaryl fused to the pyridine ring. In some embodiments, the pyridinering is connected to J at the C3 position (i.e., a 3-pyridyl ring). Insome embodiments the pyridine ring may be further substituted at the C2position or at the C6 position or both. In some embodiments, thepyridine ring is substituted with a methyl group or a trifluoromethylgroup or both.

In certain embodiments, G represents a phenyl or piperidine ring.

In certain embodiments, J is absent.

In certain embodiments, R₂ represents from 1-4 substituents selectedfrom halogen, cyano, nitro, alkoxy, amino, acylamino (e.g.,R₈—C(═O)NH—), a substituted or unsubstituted cycloalkyl, heterocyclyl,aryl, or heteroaryl fused to G, and substituted or unsubstituted loweralkyl.

In certain embodiments, R₃ includes a substituent, such as a substitutedor unsubstituted alkyl or a halogen, at a position para either to X orto the ring including A.

In certain embodiments, the subject antagonists can be chosen on thebasis of their selectively for the hedgehog pathway. This selectivitycan be for the hedgehog pathway versus other pathways, or forselectivity between particular hedgehog pathways, e.g., ptc-1, ptc-2,etc.

In certain preferred embodiments, the subject inhibitors inhibit ptcloss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function mediated signal transduction with an ED₅₀ of 1 mM orless, more preferably of 1 μM or less, and even more preferably of 1 nMor less. Similarly, in certain preferred embodiments, the subjectinhibitors inhibit activity of the hedgehog pathway with a K_(i) lessthan 10 nM, preferably less than 1 nM, even more preferably less than0.1 nM.

In particular embodiments, the small molecule is chosen for use becauseit is more selective for one patched isoform over the next, e.g.,10-fold, and more preferably at least 100- or even 1000-fold moreselective for one patched pathway (ptc-1, ptc-2) over another.

In certain embodiments, a compound which is an antagonist of thehedgehog pathway is chosen to selectively antagonize hedgehog activityover protein kinases other than PKA, such as PKC, e.g., the compoundmodulates the activity of the hedgehog pathway at least an order ofmagnitude more strongly than it modulates the activity of anotherprotein kinase, preferably at least two orders of magnitude morestrongly, even more preferably at least three orders of magnitude morestrongly. Thus, for example, a preferred inhibitor of the hedgehogpathway may inhibit hedgehog activity with a K_(i) at least an order ofmagnitude lower than its K_(i) for inhibition of PKC, preferably atleast two orders of magnitude lower, even more preferably at least threeorders of magnitude lower. In certain embodiments, the K_(i) for PKAinhibition is less than 10 nM, preferably less than 1 nM, even morepreferably less than 0.1 nM.

It is readily appreciated that any and all combinations of featurespresented in embodiments disclosed above are contemplated to be withinthe scope of the present invention.

IV. Exemplary Applications of Method and Compositions

Another aspect of the present invention relates to a method ofmodulating a differentiated state, survival, and/or proliferation of acell having a ptc loss-of-function, hedgehog gain-of-function, orsmoothened gain-of-function, by contacting the cells with a hedgehogantagonist according to the subject method and as the circumstances maywarrant.

For instance, it is contemplated by the invention that, in light of thefindings of an apparently broad involvement of hedgehog, ptc, andsmoothened in the formation of ordered spatial arrangements ofdifferentiated tissues in vertebrates, the subject method could be usedas part of a process for generating and/or maintaining an array ofdifferent vertebrate tissue both in vitro and in vivo. The hedgehogantagonist, whether inductive or anti-inductive with respectproliferation or differentiation of a given tissue, can be, asappropriate, any of the preparations described above.

For example, the present method is applicable to cell culture techniqueswherein, whether for genetic or biochemical reasons, the cells have aptc loss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function phenotype. In vitro neuronal culture systems haveproved to be fundamental and indispensable tools for the study of neuraldevelopment, as well as the identification of neurotrophic factors suchas nerve growth factor (NGF), ciliary trophic factors (CNTF), and brainderived neurotrophic factor (BDNF). One use of the present method may bein cultures of neuronal stem cells, such as in the use of such culturesfor the generation of new neurons and glia. In such embodiments of thesubject method, the cultured cells can be contacted with a hedgehogantagonist of the present invention in order to alter the rate ofproliferation of neuronal stern cells in the culture and/or alter therate of differentiation, or to maintain the integrity of a culture ofcertain terminally differentiated neuronal cells. In an exemplaryembodiment, the subject method can be used to culture, for example,sensory neurons or, alternatively, motorneurons. Such neuronal culturescan be used as convenient assay systems as well as sources ofimplantable cells for therapeutic treatments.

According to the present invention, large numbers of non-tumorigenicneural progenitor cells can be perpetuated in vitro and their rate ofproliferation and/or differentiation can be affected by contact withhedgehog antagonists of the present invention. Generally, a method isprovided comprising the steps of isolating neural progenitor cells froman animal, perpetuating these cells in vitro or in vivo, preferably inthe presence of growth factors, and regulating the differentiation ofthese cells into particular neural phenotypes, e.g., neurons and glia,by contacting the cells with a hedgehog antagonist.

Progenitor cells are thought to be under a tonic inhibitory influencethat maintains the progenitors in a suppressed state until theirdifferentiation is required. However, recent techniques have beenprovided which permit these cells to be proliferated, and unlike neuronsthat are terminally differentiated and therefore non-dividing, they canbe produced in unlimited number and are highly suitable fortransplantation into heterologous and autologous hosts withneurodegenerative diseases.

By “progenitor” it is meant an oligopotent or multipotent stem cell thatis able to divide without limit and, under specific conditions, canproduce daughter cells that terminally differentiate such as intoneurons and glia. These cells can be used for transplantation into aheterologous or autologous host. By heterologous is meant a host otherthan the animal from which the progenitor cells were originally derived.By autologous is meant the identical host from which the cells wereoriginally derived.

Cells can be obtained from embryonic, post-natal, juvenile or adultneural tissue from any animal. By any animal is meant any multicellularanimal that contains nervous tissue. More particularly, is meant anyfish, reptile, bird, amphibian or mammal and the like. The mostpreferable donors are mammals, especially mice and humans.

In the case of a heterologous donor animal, the animal may beeuthanized, and the brain and specific area of interest removed using asterile procedure. Brain areas of particular interest include any areafrom which progenitor cells can be obtained which will serve to restorefunction to a degenerated area of the host's brain. These regionsinclude areas of the central nervous system (CNS) including the cerebralcortex, cerebellum, midbrain, brainstem, spinal cord and ventriculartissue, and areas of the peripheral nervous system (PNS) including thecarotid body and the adrenal medulla. More particularly, these areasinclude regions in the basal ganglia, preferably the striatum whichconsists of the caudate and putamen, or various cell groups such as theglobus pallidus, the subthalamic nucleus, the nucleus basalis which isfound to be degenerated in Alzheimer's Disease patients, or thesubstantia nigra pars compacta which is found to be degenerated inParkinson's Disease patients.

Human heterologous neural progenitor cells may be derived from fetaltissue obtained from elective abortion, or from a post-natal, juvenileor adult organ donor. Autologous neural tissue can be obtained bybiopsy, or from patients undergoing neurosurgery in which neural tissueis removed, in particular during epilepsy surgery, and more particularlyduring temporal lobectomies and hippocampalectomies.

Cells can be obtained from donor tissue by dissociation of individualcells from the connecting extracellular matrix of the tissue.Dissociation can be obtained using any known procedure, includingtreatment with enzymes such as trypsin, collagenase and the like, or byusing physical methods of dissociation such as with a blunt instrumentor by mincing with a scalpel to a allow outgrowth of specific cell typesfrom a tissue. Dissociation of fetal cells can be carried out in tissueculture medium, while a preferable medium for dissociation of juvenileand adult cells is artificial cerebral spinal fluid (aCSF). Regular aCSFcontains 124 mM NaCl, 5 mM KCl, 1.3 mM MgCl₂, 2 mM CaCl₂, 26 mM NaHCO₃,and 10 mM D-glucose. Low Ca²⁺ aCSF contains the same ingredients exceptfor MgCl₂ at a concentration of 3.2 mM and CaCl₂ at a concentration of0.1 mM.

Dissociated cells can be placed into any known culture medium capable ofsupporting cell growth, including MEM, DMEM, RPMI, F-12, and the like,containing supplements which are required for cellular metabolism suchas glutamine and other amino acids, vitamins, minerals and usefulproteins such as transferrin and the like. Medium may also containantibiotics to prevent contamination with yeast, bacteria and fungi suchas penicillin, streptomycin, gentamicin and the like. In some cases, themedium may contain serum derived from bovine, equine, chicken and thelike. A particularly preferable medium for cells is a mixture of DMEMand F-12.

Conditions for culturing should be close to physiological conditions.The pH of the culture media should be close to physiological pH,preferably between pH 6-8, more preferably close to pH 7, even moreparticularly about pH 7.4. Cells should be cultured at a temperatureclose to physiological temperature, preferably between 30° C.-40° C.,more preferably between 32° C.-38° C., and most preferably between 35°C.-37° C.

Cells can be grown in suspension or on a fixed substrate, butproliferation of the progenitors is preferably done in suspension togenerate large numbers of cells by formation of “neurospheres” (see, forexample, Reynolds et al. (1992) Science 255:1070-1709; and PCTPublications WO93/01275, WO94/09119, WO94/10292, and WO94/16718). In thecase of propagating (or splitting) suspension cells, flasks are shakenwell and the neurospheres allowed to settle on the bottom corner of theflask. The spheres are then transferred to a 50 ml centrifuge tube andcentrifuged at low speed. The medium is aspirated, the cells resuspendedin a small amount of medium with growth factor, and the cellsmechanically dissociated and resuspended in separate aliquots of media.

Cell suspensions in culture medium are supplemented with any growthfactor which allows for the proliferation of progenitor cells and seededin any receptacle capable of sustaining cells, though as set out above,preferably in culture flasks or roller bottles. Cells typicallyproliferate within 3-4 days in a 37° C. incubator, and proliferation canbe reinitiated at any time after that by dissociation of the cells andresuspension in fresh medium containing growth factors.

In the absence of substrate, cells lift off the floor of the flask andcontinue to proliferate in suspension forming a hollow sphere ofundifferentiated cells. After approximately 3-10 days in vitro, theproliferating clusters (neurospheres) are fed every 2-7 days, and moreparticularly every 2-4 days by gentle centrifugation and resuspension inmedium containing growth factor.

After 6-7 days in vitro, individual cells in the neurospheres can beseparated by physical dissociation of the neurospheres with a bluntinstrument, more particularly by triturating the neurospheres with apipette. Single cells from the dissociated neurospheres are suspended inculture medium containing growth factors, and differentiation of thecells can be control in culture by plating (or resuspending) the cellsin the presence of a hedgehog antagonist.

To further illustrate other uses of the subject hedgehog antagonists, itis noted that intracerebral grafting has emerged as an additionalapproach to central nervous system therapies. For example, one approachto repairing damaged brain tissues involves the transplantation of cellsfrom fetal or neonatal animals into the adult brain (Dunnett et al.(1987) J Exp Biol 123:265-289; and Freund et al. (1985) J Neurosci5:603-616). Fetal neurons from a variety of brain regions can besuccessfully incorporated into the adult brain, and such grafts canalleviate behavioral defects. For example, movement disorder induced bylesions of dopaminergic projections to the basal ganglia can beprevented by grafts of embryonic dopaminergic neurons. Complex cognitivefunctions that are impaired after lesions of the neocortex can also bepartially restored by grafts of embryonic cortical cells. The subjectmethod can be used to regulate the growth state in the culture, or wherefetal tissue is used, especially neuronal stem cells, can be used toregulate the rate of differentiation of the stem cells.

Stem cells useful in the present invention are generally known. Forexample, several neural crest cells have been identified, some of whichare multipotent and likely represent uncommitted neural crest cells, andothers of which can generate only one type of cell, such as sensoryneurons, and likely represent committed progenitor cells. The role ofhedgehog antagonists employed in the present method to culture such stemcells can be to regulate differentiation of the uncommitted progenitor,or to regulate further restriction of the developmental fate of acommitted progenitor cell towards becoming a terminally differentiatedneuronal cell. For example, the present method can be used in vitro toregulate the differentiation of neural crest cells into glial cells,schwann cells, chromaffin cells, cholinergic sympathetic orparasympathetic neurons, as well as peptidergic and serotonergicneurons. The hedgehog antagonists can be used alone, or can be used incombination with other neurotrophic factors that act to moreparticularly enhance a particular differentiation fate of the neuronalprogenitor cell.

In addition to the implantation of cells cultured in the presence of thesubject hedgehog antagonists, yet another aspect of the presentinvention concerns the therapeutic application of a hedgehog antagonistto regulate the growth state of neurons and other neuronal cells in boththe central nervous system and the peripheral nervous system. Theability of ptc, hedgehog, and smoothened to regulate neuronaldifferentiation during development of the nervous system and alsopresumably in the adult state indicates that, in certain instances, thesubject hedgehog antagonists can be expected to facilitate control ofadult neurons with regard to maintenance, functional performance, andaging of normal cells; repair and regeneration processes in chemicallyor mechanically lesioned cells; and treatment of degeneration in certainpathological conditions. In light of this understanding, the presentinvention specifically contemplates applications of the subject methodto the treatment protocol of (prevention and/or reduction of theseverity of) neurological conditions deriving from: (i) acute, subacute,or chronic injury to the nervous system, including traumatic injury,chemical injury, vascular injury and deficits (such as the ischemiaresulting from stroke), together with infectious/inflammatory andtumor-induced injury; (ii) aging of the nervous system includingAlzheimer's disease; (iii) chronic neurodegenerative diseases of thenervous system, including Parkinson's disease, Huntington's chorea,amylotrophic lateral sclerosis and the like, as well as spinocerebellardegenerations; and (iv) chronic immunological diseases of the nervoussystem or affecting the nervous system, including multiple sclerosis.

As appropriate, the subject method can also be used in generating nerveprostheses for the repair of central and peripheral nerve damage. Inparticular, where a crushed or severed axon is intubulated by use of aprosthetic device, hedgehog antagonists can be added to the prostheticdevice to regulate the rate of growth and regeneration of the dendridicprocesses. Exemplary nerve guidance channels are described in U.S. Pat.Nos. 5,092,871 and 4,955,892.

In another embodiment, the subject method can be used in the treatmentof neoplastic or hyperplastic transformations such as may occur in thecentral nervous system. For instance, the hedgehog antagonists can beutilized to cause such transformed cells to become either post-mitoticor apoptotic. The present method may, therefore, be used as part of atreatment for, e.g., malignant gliomas, meningiomas, medulloblastomas,neuroectodermal tumors, and ependymomas.

In a preferred embodiment, the subject method can be used as part of atreatment regimen for malignant medulloblastoma and other primary CNSmalignant neuroectodermal tumors.

In certain embodiments, the subject method is used as part of treatmentprogram for medulloblastoma. Medulloblastoma, a primary brain tumor, isthe most common brain tumor in children. A medulloblastoma is aprimitive neuroectodermal tumor arising in the posterior fossa. Theyaccount for approximately 25% of all pediatric brain tumors (Miller).Histologically, they are small round cell tumors commonly arranged intrue rosettes, but may display some differentiation to astrocytes,ependymal cells or neurons (Rorke; Kleihues). PNET's may arise in otherareas of the brain including the pineal gland (pineoblastoma) andcerebrum. Those arising in the supratentorial region generally fareworse than their PF counterparts.

Medulloblastoma/PNET's are known to recur anywhere in the CNS afterresection, and can even metastasize to bone. Pretreatment evaluationshould therefore include an examination of the spinal cord to excludethe possibility of “dropped metastases”. Gadolinium-enhanced MRI haslargely replaced myelography for this purpose, and CSF cytology isobtained postoperatively as a routine procedure.

In other embodiments, the subject method is used as part of treatmentprogram for ependymomas. Ependymomas account for approximately 10% ofthe pediatric brain tumors in children. Grossly, they are tumors thatarise from the ependymal lining of the ventricles and microscopicallyform rosettes, canals, and perivascular rosettes. In the CHOP series of51 children reported with ependymomas, ¾ were histologically benign.Approximately ⅔ arose from the region of the 4th ventricle. One thirdpresented in the supratentorial region. Age at presentation peaksbetween birth and 4 years, as demonstrated by SEER data as well as datafrom CHOP. The median age is about 5 years. Because so many childrenwith this disease are babies, they often require multimodal therapy.

Yet another aspect of the present invention concerns the observation inthe art that ptc, hedgehog, and/or smoothened are involved inmorphogenic signals involved in other vertebrate organogenic pathways inaddition to neuronal differentiation as described above, having apparentroles in other endodermal patterning, as well as both mesodermal andendodermal differentiation processes. Thus, it is contemplated by theinvention that compositions comprising hedgehog antagonists can also beutilized for both cell culture and therapeutic methods involvinggeneration and maintenance of non-neuronal tissue.

In one embodiment, the present invention makes use of the discovery thatptc, hedgehog, and smoothened are apparently involved in controlling thedevelopment of stem cells responsible for formation of the digestivetract, liver, lungs, and other organs which derive from the primitivegut. Shh serves as an inductive signal from the endoderm to themesoderm, which is critical to gut morphogenesis. Therefore, forexample, hedgehog antagonists of the instant method can be employed forregulating the development and maintenance of an artificial liver thatcan have multiple metabolic functions of a normal liver. In an exemplaryembodiment, the subject method can be used to regulate the proliferationand differentiation of digestive tube stem cells to form hepatocytecultures which can be used to populate extracellular matrices, or whichcan be encapsulated in biocompatible polymers, to form both implantableand extracorporeal artificial livers.

In another embodiment, therapeutic compositions of hedgehog antagonistscan be utilized in conjunction with transplantation of such artificiallivers, as well as embryonic liver structures, to regulate uptake ofintraperitoneal implantation, vascularization, and in vivodifferentiation and maintenance of the engrafted liver tissue.

In yet another embodiment, the subject method can be employedtherapeutically to regulate such organs after physical, chemical orpathological insult. For instance, therapeutic compositions comprisinghedgehog antagonists can be utilized in liver repair subsequent to apartial hepatectomy.

The generation of the pancreas and small intestine from the embryonicgut depends on intercellular signalling between the endodermal andmesodermal cells of the gut. In particular, the differentiation ofintestinal mesoderm into smooth muscle has been suggested to depend onsignals from adjacent endodermal cells. One candidate mediator ofendodermally derived signals in the embryonic hindgut is Sonic hedgehog.See, for example, Apelqvist et al. (1997) Curr Biol 7:801-4. The Shhgene is expressed throughout the embryonic gut endoderm with theexception of the pancreatic bud endoderm, which instead expresses highlevels of the homeodomain protein Ipf1/Pdx1 (insulin promoter factor1/pancreatic and duodenal homeobox 1), an essential regulator of earlypancreatic development. Apelqvist et al., supra, have examined whetherthe differential expression of Shh in the embryonic gut tube controlsthe differentiation of the surrounding mesoderm into specialisedmesoderm derivatives of the small intestine and pancreas. To test this,they used the promoter of the Ipf1/Pdx1 gene to selectively express Shhin the developing pancreatic epithelium. In Ipf1/Pdx1-Shh transgenicmice, the pancreatic mesoderm developed into smooth muscle andinterstitial cells of Cajal, characteristic of the intestine, ratherthan into pancreatic mesenchyme and spleen. Also, pancreatic explantsexposed to Shh underwent a similar program of intestinaldifferentiation. These results provide evidence that the differentialexpression of endodermally derived Shh controls the fate of adjacentmesoderm at different regions of the gut tube.

In the context of the present invention, it is contemplated thereforethat the subject hedgehog antagonists can be used to control or regulatethe proliferation and/or differentiation of pancreatic tissue both invivo and in vitro.

There are a wide variety of pathological cell proliferative anddifferentiative conditions for which the inhibitors of the presentinvention may provide therapeutic benefits, with the general strategybeing, for example, the correction of aberrant insulin expression, ormodulation of differentiation. More generally, however, the presentinvention relates to a method of inducing and/or maintaining adifferentiated state, enhancing survival and/or affecting proliferationof pancreatic cells, by contacting the cells with the subjectinhibitors. For instance, it is contemplated by the invention that, inlight of the apparent involvement of ptc, hedgehog, and smoothened inthe formation of ordered spatial arrangements of pancreatic tissues, thesubject method could be used as part of a technique to generate and/ormaintain such tissue both in vitro and in vivo. For instance, modulationof the function of hedgehog can be employed in both cell culture andtherapeutic methods involving generation and maintenance β-cells andpossibly also for non-pancreatic tissue, such as in controlling thedevelopment and maintenance of tissue from the digestive tract, spleen,lungs, urogenital organs (e.g., bladder), and other organs which derivefrom the primitive gut.

In an exemplary embodiment, the present method can be used in thetreatment of hyperplastic and neoplastic disorders effecting pancreatictissue, particularly those characterized by aberrant proliferation ofpancreatic cells. For instance, pancreatic cancers are marked byabnormal proliferation of pancreatic cells that can result inalterations of insulin secretory capacity of the pancreas. For instance,certain pancreatic hyperplasias, such as pancreatic carcinomas, canresult in hypoinsulinemia due to dysfunction of α-cells or decreasedislet cell mass. To the extent that aberrant ptc, hedgehog, andsmoothened signaling may be indicated in disease progression, thesubject inhibitors, can be used to enhance regeneration of the tissueafter anti-tumor therapy.

Moreover, manipulation of hedgehog signaling properties at differentpoints may be useful as part of a strategy for reshaping/repairingpancreatic tissue both in vivo and in vitro. In one embodiment, thepresent invention makes use of the apparent involvement of ptc,hedgehog, and smoothened in regulating the development of pancreatictissue. In general, the subject method can be employed therapeuticallyto regulate the pancreas after physical, chemical or pathologicalinsult. In yet another embodiment, the subject method can be applied tocell culture techniques, and in particular, may be employed to enhancethe initial generation of prosthetic pancreatic tissue devices.Manipulation of proliferation and differentiation of pancreatic tissue,for example, by altering hedgehog activity, can provide a means for morecarefully controlling the characteristics of a cultured tissue. In anexemplary embodiment, the subject method can be used to augmentproduction of prosthetic devices which require β-islet cells, such asmay be used in the encapsulation devices described in, for example, theAebischer et al. U.S. Pat. No. 4,892,538, the Aebischer et al. U.S. Pat.No. 5,106,627, the Lim U.S. Pat. No. 4,391,909, and the Sefton U.S. Pat.No. 4,353,888. Early progenitor cells to the pancreatic islets aremultipotential, and apparently coactivate all the islet-specific genesfrom the time they first appear. As development proceeds, expression ofislet-specific hormones, such as insulin, becomes restricted to thepattern of expression characteristic of mature islet cells. Thephenotype of mature islet cells, however, is not stable in culture, asreappearance of embryonal traits in mature β-cells can be observed. Byutilizing the subject hedgehog antagonists, the differentiation path orproliferative index of the cells can be regulated.

Furthermore, manipulation of the differentiative state of pancreatictissue can be utilized in conjunction with transplantation of artificialpancreas so as to promote implantation, vascularization, and in vivodifferentiation and maintenance of the engrafted tissue. For instance,manipulation of hedgehog function to affect tissue differentiation canbe utilized as a means of maintaining graft viability.

Bellusci et al. (1997) Development 124:53 report that Sonic hedgehogregulates lung mesenchymal cell proliferation in vivo. Accordingly, thepresent method can be used to regulate regeneration of lung tissue,e.g., in the treatment of emphysema and respiratory distress syndrome.

Fujita et al. (1997) Biochem Biophys Res Commun 238:658 reported thatSonic hedgehog is expressed in human lung squamous carcinoma andadenocarcinoma cells. The expression of Sonic hedgehog was also detectedin the human lung squamous carcinoma tissues, but not in the normal lungtissue of the same patient. They also observed that Sonic hedgehogstimulates the incorporation of BrdU into the carcinoma cells andstimulates their cell growth, while anti-Shh-N inhibited their cellgrowth. These results suggest that a ptc, hedgehogs and/or smoothened isinvolved in the cell growth of such transformed lung tissue andtherefore indicates that the subject method can be used as part of atreatment of lung carcinoma and adenocarcinomas, and other proliferativedisorders involving the lung epithelia.

Many other tumors may, based on evidence such as involvement of thehedgehog pathway in these tumors, or detected expression of hedgehog orits receptor in these tissues during development, be affected bytreatment with the subject compounds. Such tumors include, but are by nomeans limited to, tumors related to Gorlin's syndrome (e.g., basal cellcarcinoma, medulloblastoma, meningioma, etc.), tumors evidenced in pctknock-out mice (e.g., hemangioma, rhabdomyosarcoma, etc.), tumorsresulting from gli-1 amplification (e.g., glioblastoma, sarcoma, etc.),tumors connected with TRC8, a ptc homolog (e.g., renal carcinoma,thyroid carcinoma, etc.), Ext-1-related tumors (e.g., bone cancer,etc.), Shh-induced tumors (e.g., lung cancer, chondrosarcomas, etc.),and other tumors (e.g., breast cancer, urogenital cancer (e.g., kidney,bladder, ureter, prostate, etc.), adrenal cancer, gastrointestinalcancer (e.g., stomach, intestine, etc.), etc.).

In still another embodiment of the present invention, compositionscomprising hedgehog antagonists can be used in the in vitro generationof skeletal tissue, such as from skeletogenic stem cells, as well as thein vivo treatment of skeletal tissue deficiencies. The present inventionparticularly contemplates the use of hedgehog antagonists to regulatethe rate of chondrogenesis and/or osteogenesis. By “skeletal tissuedeficiency”, it is meant a deficiency in bone or other skeletalconnective tissue at any site where it is desired to restore the bone orconnective tissue, no matter how the deficiency originated, e.g. whetheras a result of surgical intervention, removal of tumor, ulceration,implant, fracture, or other traumatic or degenerative conditions.

For instance, the method of the present invention can be used as part ofa regimen for restoring cartilage function to a connective tissue. Suchmethods are useful in, for example, the repair of defects or lesions incartilage tissue which is the result of degenerative wear such as thatwhich results in arthritis, as well as other mechanical derangementswhich may be caused by trauma to the tissue, such as a displacement oftorn meniscus tissue, meniscectomy, a laxation of a joint by a tornligament, malignment of joints, bone fracture, or by hereditary disease.The present reparative method is also useful for remodeling cartilagematrix, such as in plastic or reconstructive surgery, as well asperiodontal surgery. The present method may also be applied to improvinga previous reparative procedure, for example, following surgical repairof a meniscus, ligament, or cartilage. Furthermore, it may prevent theonset or exacerbation of degenerative disease if applied early enoughafter trauma.

In one embodiment of the present invention, the subject method comprisestreating the afflicted connective tissue with a therapeuticallysufficient amount of a hedgehog antagonist, particularly an antagonistselective for Indian hedgehog signal transduction, to regulate acartilage repair response in the connective tissue by managing the rateof differentiation and/or proliferation of chondrocytes embedded in thetissue. Such connective tissues as articular cartilage, interarticularcartilage (menisci), costal cartilage (connecting the true ribs and thesternum), ligaments, and tendons are particularly amenable to treatmentin reconstructive and/or regenerative therapies using the subjectmethod. As used herein, regenerative therapies include treatment ofdegenerative states which have progressed to the point of whichimpairment of the tissue is obviously manifest, as well as preventivetreatments of tissue where degeneration is in its earliest stages orimminent.

In an illustrative embodiment, the subject method can be used as part ofa therapeutic intervention in the treatment of cartilage of adiarthroidal joint, such as a knee, an ankle, an elbow, a hip, a wrist,a knuckle of either a finger or toe, or a tempomandibular joint. Thetreatment can be directed to the meniscus of the joint, to the articularcartilage of the joint, or both. To further illustrate, the subjectmethod can be used to treat a degenerative disorder of a knee, such aswhich might be the result of traumatic injury (e.g., a sports injury orexcessive wear) or osteoarthritis. The subject antagonists may beadministered as an injection into the joint with, for instance, anarthroscopic needle. In some instances, the injected agent can be in theform of a hydrogel or other slow release vehicle described above inorder to permit a more extended and regular contact of the agent withthe treated tissue.

The present invention further contemplates the use of the subject methodin the field of cartilage transplantation and prosthetic devicetherapies. However, problems arise, for instance, because thecharacteristics of cartilage and fibrocartilage varies between differenttissue: such as between articular, meniscal cartilage, ligaments, andtendons, between the two ends of the same ligament or tendon, andbetween the superficial and deep parts of the tissue. The zonalarrangement of these tissues may reflect a gradual change in mechanicalproperties, and failure occurs when implanted tissue, which has notdifferentiated under those conditions, lacks the ability toappropriately respond. For instance, when meniscal cartilage is used torepair anterior cruciate ligaments, the tissue undergoes a metaplasia topure fibrous tissue. By regulating the rate of chondrogenesis, thesubject method can be used to particularly address this problem, byhelping to adaptively control the implanted cells in the new environmentand effectively resemble hypertrophic chondrocytes of an earlierdevelopmental stage of the tissue.

In similar fashion, the subject method can be applied to enhancing boththe generation of prosthetic cartilage devices and to theirimplantation. The need for improved treatment has motivated researchaimed at creating new cartilage that is based oncollagen-glycosaminoglycan templates (Stone et al. (1990) Clin OrthopRelat Red 252:129), isolated chondrocytes (Grande et al. (1989) J OrthopRes 7:208; and Takigawa et al. (1987) Bone Miner 2:449), andchondrocytes attached to natural or synthetic polymers (Walitani et al.(1989) J Bone Jt Surg 71B:74; Vacanti et al. (1991) Plast Reconstr Surg88:753; von Schroeder et al. (1991) J Biomed Mater Res 25:329; Freed etal. (1993) J Biomed Mater Res 27:11; and the Vacanti et al. U.S. Pat.No. 5,041,138). For example, chondrocytes can be grown in culture onbiodegradable, biocompatible highly porous scaffolds formed frompolymers such as polyglycolic acid, polylactic acid, agarose gel, orother polymers that degrade over time as function of hydrolysis of thepolymer backbone into innocuous monomers. The matrices are designed toallow adequate nutrient and gas exchange to the cells until engraftmentoccurs. The cells can be cultured in vitro until adequate cell volumeand density has developed for the cells to be implanted. One advantageof the matrices is that they can be cast or molded into a desired shapeon an individual basis, so that the final product closely resembles thepatient's own ear or nose (by way of example), or flexible matrices canbe used which allow for manipulation at the time of implantation, as ina joint.

In one embodiment of the subject method, the implants are contacted witha hedgehog antagonist during certain stages of the culturing process inorder to manage the rate of differentiation of chondrocytes and theformation of hypertrophic chrondrocytes in the culture.

In another embodiment, the implanted device is treated with a hedgehogantagonist in order to actively remodel the implanted matrix and to makeit more suitable for its intended function. As set out above withrespect to tissue transplants, the artificial transplants suffer fromthe same deficiency of not being derived in a setting which iscomparable to the actual mechanical environment in which the matrix isimplanted. The ability to regulate the chondrocytes in the matrix by thesubject method can allow the implant to acquire characteristics similarto the tissue for which it is intended to replace.

In yet another embodiment, the subject method is used to enhanceattachment of prosthetic devices. To illustrate, the subject method canbe used in the implantation of a periodontal prosthesis, wherein thetreatment of the surrounding connective tissue stimulates formation ofperiodontal ligament about the prosthesis.

In still further embodiments, the subject method can be employed as partof a regimen for the generation of bone (osteogenesis) at a site in theanimal where such skeletal tissue is deficient. Indian hedgehog isparticularly associated with the hypertrophic chondrocytes that areultimately replaced by osteoblasts. For instance, administration of ahedgehog antagonist of the present invention can be employed as part ofa method for regulating the rate of bone loss in a subject. For example,preparations comprising hedgehog antagonists can be employed, forexample, to control endochondral ossification in the formation of a“model” for ossification.

In yet another embodiment of the present invention, a hedgehogantagonist can be used to regulate spermatogenesis. The hedgehogproteins, particularly Dhh, have been shown to be involved in thedifferentiation and/or proliferation and maintenance of testicular germcells. Dhh expression is initiated in Sertoli cell precursors shortlyafter the activation of Sry (testicular determining gene) and persistsin the testis into the adult. Males are viable but infertile, owing to acomplete absence of mature sperm. Examination of the developing testisin different genetic backgrounds suggests that Dhh regulates both earlyand late stages of spermatogenesis. Bitgood et al. (1996) Curr Biol6:298. In a preferred embodiment, the hedgehog antagonist can be used asa contraceptive. In similar fashion, hedgehog antagonists of the subjectmethod are potentially useful for modulating normal ovarian function.

The subject method also has wide applicability to the treatment orprophylaxis of disorders afflicting epithelial tissue, as well as incosmetic uses. In general, the method can be characterized as includinga step of administering to an animal an amount of a hedgehog antagonisteffective to alter the growth state of a treated epithelial tissue. Themode of administration and dosage regimens will vary depending on theepithelial tissue(s) to be treated. For example, topical formulationswill be preferred where the treated tissue is epidermal tissue, such asdermal or mucosal tissues.

A method that “promotes the healing of a wound” results in the woundhealing more quickly as a result of the treatment than a similar woundheals in the absence of the treatment. “Promotion of wound healing” canalso mean that the method regulates the proliferation and/or growth of,inter alia, keratinocytes, or that the wound heals with less scarring,less wound contraction, less collagen deposition and more superficialsurface area. In certain instances, “promotion of wound healing” canalso mean that certain methods of wound healing have improved successrates, (e.g., the take rates of skin grafts,) when used together withthe method of the present invention.

Despite significant progress in reconstructive surgical techniques,scarring can be an important obstacle in regaining normal function andappearance of healed skin. This is particularly true when pathologicscarring such as keloids or hypertrophic scars of the hands or facecauses functional disability or physical deformity. In the severestcircumstances, such scarring may precipitate psychosocial distress and alife of economic deprivation. Wound repair includes the stages ofhemostasis, inflammation, proliferation, and remodeling. Theproliferative stage involves multiplication of fibroblasts andendothelial and epithelial cells. Through the use of the subject method,the rate of proliferation of epithelial cells in and proximal to thewound can be controlled in order to accelerate closure of the woundand/or minimize the formation of scar tissue.

The present treatment can also be effective as part of a therapeuticregimen for treating oral and paraoral ulcers, e.g. resulting fromradiation and/or chemotherapy. Such ulcers commonly develop within daysafter chemotherapy or radiation therapy. These ulcers usually begin assmall, painful irregularly shaped lesions usually covered by a delicategray necrotic membrane and surrounded by inflammatory tissue. In manyinstances, lack of treatment results in proliferation of tissue aroundthe periphery of the lesion on an inflammatory basis. For instance, theepithelium bordering the ulcer usually demonstrates proliferativeactivity, resulting in loss of continuity of surface epithelium. Theselesions, because of their size and loss of epithelial integrity, disposethe body to potential secondary infection. Routine ingestion of food andwater becomes a very painful event and, if the ulcers proliferatethroughout the alimentary canal, diarrhea usually is evident with allits complicating factors. According to the present invention, atreatment for such ulcers which includes application of a hedgehogantagonist can reduce the abnormal proliferation and differentiation ofthe affected epithelium, helping to reduce the severity of subsequentinflammatory events.

The subject method and compositions can also be used to treat woundsresulting from dermatological diseases, such as lesions resulting fromautoimmune disorders such as psoriasis. Atopic dermititis refers to skintrauma resulting from allergies associated with an immune responsecaused by allergens such as pollens, foods, dander, insect venoms andplant toxins.

In other embodiments, antiproliferative preparations of hedgehogantagonists can be used to inhibit lens epithelial cell proliferation toprevent post-operative complications of extracapsular cataractextraction. Cataract is an intractable eye disease and various studieson a treatment of cataract have been made. But at present, the treatmentof cataract is attained by surgical operations. Cataract surgery hasbeen applied for a long time and various operative methods have beenexamined. Extracapsular lens extraction has become the method of choicefor removing cataracts. The major medical advantages of this techniqueover intracapsular extraction are lower incidence of aphakic cystoidmacular edema and retinal detachment. Extracapsular extraction is alsorequired for implantation of posterior chamber type intraocular lensesthat are now considered to be the lenses of choice in most cases.

However, a disadvantage of extracapsular cataract extraction is the highincidence of posterior lens capsule opacification, often calledafter-cataract, which can occur in up to 50% of cases within three yearsafter surgery. After-cataract is caused by proliferation of equatorialand anterior capsule lens epithelial cells that remain afterextracapsular lens extraction. These cells proliferate to causeSommerling rings, and along with fibroblasts that also deposit and occuron the posterior capsule, cause opacification of the posterior capsule,which interferes with vision. Prevention of after-cataract would bepreferable to treatment. To inhibit secondary cataract formation, thesubject method provides a means for inhibiting proliferation of theremaining lens epithelial cells. For example, such cells can be inducedto remain quiescent by instilling a solution containing an hedgehogantagonist preparation into the anterior chamber of the eye after lensremoval. Furthermore, the solution can be osmotically balanced toprovide minimal effective dosage when instilled into the anteriorchamber of the eye, thereby inhibiting subcapsular epithelial growthwith some specificity.

The subject method can also be used in the treatment of comeopathiesmarked by corneal epithelial cell proliferation, as for example inocular epithelial disorders such as epithelial downgrowth or squamouscell carcinomas of the ocular surface.

Levine et al. (1997) J Neurosci 17:6277 show that hedgehog proteins canregulate mitogenesis and photoreceptor differentiation in the vertebrateretina, and Ihh is a candidate factor from the pigmented epithelium topromote retinal progenitor proliferation and photoreceptordifferentiation. Likewise, Jensen et al. (1997) Development 124:363demonstrated that treatment of cultures of perinatal mouse retinal cellswith the amino-terminal fragment of Sonic hedgehog protein results in anincrease in the proportion of cells that incorporate bromodeoxuridine,in total cell numbers, and in rod photoreceptors, amacrine cells andMuller glial cells, suggesting that Sonic hedgehog promotes theproliferation of retinal precursor cells. Thus, the subject method canbe used in the treatment of proliferative diseases of retinal cells andregulate photoreceptor differentiation.

Yet another aspect of the present invention relates to the use of thesubject method to control hair growth. Hair is basically composed ofkeratin, a tough and insoluble protein; its chief strength lies in itsdisulphide bond of cystine. Each individual hair comprises a cylindricalshaft and a root, and is contained in a follicle, a flask-likedepression in the skin. The bottom of the follicle contains afinger-like projection termed the papilla, which consists of connectivetissue from which hair grows, and through which blood vessels supply thecells with nourishment. The shaft is the part that extends outwards fromthe skin surface, whilst the root has been described as the buried partof the hair. The base of the root expands into the hair bulb, whichrests upon the papilla. Cells from which the hair is produced grow inthe bulb of the follicle; they are extruded in the form of fibers as thecells proliferate in the follicle. Hair “growth” refers to the formationand elongation of the hair fiber by the dividing cells.

As is well known in the art, the common hair cycle is divided into threestages: anagen, catagen and telogen. During the active phase (anagen),the epidermal stem cells of the dermal papilla divide rapidly. Daughtercells move upward and differentiate to form the concentric layers of thehair itself. The transitional stage, catagen, is marked by the cessationof mitosis of the stem cells in the follicle. The resting stage is knownas telogen, where the hair is retained within the scalp for severalweeks before an emerging new hair developing below it dislodges thetelogen-phase shaft from its follicle. From this model it has becomeclear that the larger the pool of dividing stem cells that differentiateinto hair cells, the more hair growth occurs. Accordingly, methods forincreasing or reducing hair growth can be carried out by potentiating orinhibiting, respectively, the proliferation of these stem cells.

In certain embodiments, the subject method can be employed as a way ofreducing the growth of human hair as opposed to its conventional removalby cutting, shaving, or depilation. For instance, the present method canbe used in the treatment of trichosis characterized by abnormally rapidor dense growth of hair, e.g. hypertrichosis. In an exemplaryembodiment, hedgehog antagonists can be used to manage hirsutism, adisorder marked by abnormal hairiness. The subject method can alsoprovide a process for extending the duration of depilation.

Moreover, because a hedgehog antagonist will often be cytostatic toepithelial cells, rather than cytotoxic, such agents can be used toprotect hair follicle cells from cytotoxic agents that requireprogression into S-phase of the cell-cycle for efficacy, e.g.radiation-induced death. Treatment by the subject method can provideprotection by causing the hair follicle cells to become quiescent, e.g.,by inhibiting the cells from entering S phase, and thereby preventingthe follicle cells from undergoing mitotic catastrophe or programmedcell death. For instance, hedgehog antagonists can be used for patientsundergoing chemo- or radiation-therapies that ordinarily result in hairloss. By inhibiting cell-cycle progression during such therapies, thesubject treatment can protect hair follicle cells from death that mightotherwise result from activation of cell death programs. After thetherapy has concluded, the instant method can also be removed withconcommitant relief of the inhibition of follicle cell proliferation.

The subject method can also be used in the treatment of folliculitis,such as folliculitis decalvans, folliculitis ulerythematosa reticulataor keloid folliculitis. For example, a cosmetic preparation of anhedgehog antagonist can be applied topically in the treatment ofpseudofolliculitis, a chronic disorder occurring most often in thesubmandibular region of the neck and associated with shaving, thecharacteristic lesions of which are erythematous papules and pustulescontaining buried hairs.

In another aspect of the invention, the subject method can be used toinduce differentiation and/or inhibit proliferation of epitheliallyderived tissue. Such forms of these molecules can provide a basis fordifferentiation therapy for the treatment of hyperplastic and/orneoplastic conditions involving epithelial tissue. For example, suchpreparations can be used for the treatment of cutaneous diseases inwhich there is abnormal proliferation or growth of cells of the skin.

For instance, the pharmaceutical preparations of the invention areintended for the treatment of hyperplastic epidermal conditions, such askeratosis, as well as for the treatment of neoplastic epidermalconditions such as those characterized by a high proliferation rate forvarious skin cancers, as for example basal cell carcinoma or squamouscell carcinoma. The subject method can also be used in the treatment ofautoimmune diseases affecting the skin, in particular, of dermatologicaldiseases involving morbid proliferation and/or keratinization of theepidermis, as for example, caused by psoriasis or atopic dermatosis.

Many common diseases of the skin, such as psoriasis, squamous cellcarcinoma, keratoacanthoma and actinic keratosis are characterized bylocalized abnormal proliferation and growth. For example, in psoriasis,which is characterized by scaly, red, elevated plaques on the skin, thekeratinocytes are known to proliferate much more rapidly than normal andto differentiate less completely.

In one embodiment, the preparations of the present invention aresuitable for the treatment of dermatological ailments linked tokeratinization disorders causing abnormal proliferation of skin cells,which disorders may be marked by either inflammatory or non-inflammatorycomponents. To illustrate, therapeutic preparations of a hedgehogantagonist, e.g., which promotes quiescense or differentiation can beused to treat varying forms of psoriasis, be they cutaneous, mucosal orungual. Psoriasis, as described above, is typically characterized byepidermal keratinocytes that display marked proliferative activation anddifferentiation along a “regenerative” pathway. Treatment with anantiproliferative embodiment of the subject method can be used toreverse the pathological epidermal activiation and can provide a basisfor sustained remission of the disease.

A variety of other keratotic lesions are also candidates for treatmentwith the subject method. Actinic keratoses, for example, are superficialinflammatory premalignant tumors arising on sun-exposed and irradiatedskin. The lesions are erythematous to brown with variable scaling.Current therapies include excisional and cryosurgery. These treatmentsare painful, however, and often produce cosmetically unacceptablescarring. Accordingly, treatment of keratosis, such as actinickeratosis, can include application, preferably topical, of a hedgehogantagonist composition in amounts sufficient to inhibithyperproliferation of epidermal/epidermoid cells of the lesion.

Acne represents yet another dermatologic ailment which may be treated bythe subject method. Acne vulgaris, for instance, is a multifactorialdisease most commonly occurring in teenagers and young adults, and ischaracterized by the appearance of inflammatory and noninflammatorylesions on the face and upper trunk. The basic defect which gives riseto acne vulgaris is hypercomification of the duct of a hyperactivesebaceous gland. Hypercomification blocks the normal mobility of skinand follicle microorganisms, and in so doing, stimulates the release oflipases by Propinobacterium acnes and Staphylococcus epidermidisbacteria and Pitrosporum ovale, a yeast. Treatment with anantiproliferative hedgehog antagonist, particularly topicalpreparations, may be useful for preventing the transitional features ofthe ducts, e.g. hypercomification, which lead to lesion formation. Thesubject treatment may further include, for example, antibiotics,retinoids and antiandrogens.

The present invention also provides a method for treating various formsof dermatitis. Dermatitis is a descriptive term referring to poorlydemarcated lesions that are either pruritic, erythematous, scaley,blistered, weeping, fissured or crusted. These lesions arise from any ofa wide variety of causes. The most common types of dermatitis areatopic, contact and diaper dermatitis. For instance, seborrheicdermatitis is a chronic, usually pruritic, dermatitis with erythema,dry, moist, or greasy scaling, and yellow crusted patches on variousareas, especially the scalp, with exfoliation of an excessive amount ofdry scales. The subject method can also be used in the treatment ofstasis dermatitis, an often chronic, usually eczematous dermatitis.Actinic dermatitis is dermatitis that due to exposure to actinicradiation such as that from the sun, ultraviolet waves or x- orgamma-radiation. According to the present invention, the subject methodcan be used in the treatment and/or prevention of certain symptoms ofdermatitis caused by unwanted proliferation of epithelial cells. Suchtherapies for these various forms of dermatitis can also include topicaland systemic corticosteroids, antipuritics, and antibiotics.

For example, it is contemplated that the subject method could be used toinhibit angiogenesis. Hedgehog is known to stimulate angiogenesis.Matrigel plugs impregnated with hedgehog protein and inserted into miceevince substantial neovascularization, whereas Matrigel plugs notcarrying hedgehog show comparatively little vascularization. Hedgehogprotein is also capable of increasing vascularization of the normallyavascular mouse cornea. The ptc-1 gene is expressed in normal vasculartissues, including the endothelial cells of the aorta, vascular smoothmuscle cells, adventitial fibroblasts of the aorta, the coronaryvasculature and cardiomyocytes of the atria and ventricles. Thesetissues are also sensitive to hedgehog protein. Treatment with exogenoushedgehog causes upregulation of ptc-1 expression. In addition, hedgehogproteins stimulate proliferation of vascular smooth muscle cells invivo. Hedgehog proteins also cause fibroblasts to increase expression ofangiogenic growth factors such as VEGF, bFGF, Ang-1 and Ang-2. Lastly,hedgehog proteins are known to stimulate recovery from ischemic injuryand stimulate formation of collateral vessels.

Given that hedgehog promotes angiogenesis, hedgehog antagonists areexpected to act as angiogenesis inhibitors, particularly in situationswhere some level of hedgehog signaling is necessary for angiogenesis.

Angiogenesis is fundamental to many disorders. Persistent, unregulatedangiogenesis occurs in a range of disease states, tumor metastases andabnormal growths by endothelial cells. The vasculature created as aresult of angiogenic processes supports the pathological damage seen inthese conditions. The diverse pathological states created due tounregulated angiogenesis have been grouped together as angiogenicdependent or angiogenic associated diseases. Therapies directed atcontrol of the angiogenic processes could lead to the abrogation ormitigation of these diseases.

Diseases caused by, supported by or associated with angiogenesis includeocular neovascular disease, age-related macular degeneration, diabeticretinopathy, retinopathy of prematurity, corneal graft rejection,neovascular glaucoma, retrolerital fibroplasia, epidemickeratoconjunctivitis, Vitamin A deficiency, contact lens overwear,atopic keratitis, superior limbic keratitis, pterygium keratitis sicca,Sjogren's, acne rosacea, phylectenulosis, syphilis, Mycobacteriainfections, lipid degeneration, chemical burns, bacterial ulcers, fungalulcers, Herpes simplex infections, Herpes zoster infections, protozoaninfections, Kaposi sarcoma, Mooren ulcer, Terrien's marginaldegeneration, marginal keratolysis, rheumatoid arthritis, systemiclupus, polyarteritis, trauma, Wegeners sarcoidosis, Scleritis, StevensJohnson disease, periphigoid radial keratotomy, corneal graph rejection,rheumatoid arthritis, osteoarthritis chronic inflammation (eg.,ulcerative colitis or Crohn's disease), hemangioma, Osler-Weber-Rendudisease, and hereditary hemorrhagic telangiectasia.

In addition, angiogenesis plays a critical role in cancer. A tumorcannot expand without a blood supply to provide nutrients and removecellular wastes. Tumors in which angiogenesis is important include solidtumors such as rhabdomyosarcomas, retinoblastoma, Ewing sarcoma,neuroblastoma, and osteosarcoma, and benign tumors such as acousticneuroma, neurofibroma, trachoma and pyogenic granulomas. Angiogenicfactors have been found associated with several solid tumors. Preventionof angiogenesis could halt the growth of these tumors and the resultantdamage to the animal due to the presence of the tumor. Angiogenesis isalso associated with blood-born tumors such as leukemias, any of variousacute or chronic neoplastic diseases of the bone marrow in whichunrestrained proliferation of white blood cells occurs, usuallyaccompanied by anemia, impaired blood clotting, and enlargement of thelymph nodes, liver, and spleen. It is believed that angiogenesis plays arole in the abnormalities in the bone marrow that give rise toleukemia-like tumors.

In addition to tumor growth, angiogenesis is important in metastasis.Initially, angiogenesis is important is in the vascularization of thetumor which allows cancerous cells to enter the blood stream and tocirculate throughout the body. After the tumor cells have left theprimary site, and have settled into the secondary, metastasis site,angiogenesis must occur before the new tumor can grow and expand.Therefore, prevention of angiogenesis could lead to the prevention ofmetastasis of tumors and possibly contain the neoplastic growth at theprimary site.

Angiogenesis is also involved in normal physiological processes such asreproduction and wound healing. Angiogenesis is an important step inovulation and also in implantation of the blastula after fertilization.Prevention of angiogenesis could be used to induce amenorrhea, to blockovulation or to prevent implantation by the blastula.

It is anticipated that the invention will be useful for the treatmentand/or prevention of respiratory distress syndrome or other disordersresulting from inappropriate lung surface tension. Respiratory distresssyndrome results from insufficient surfactant in the alveolae of thelungs. The lungs of vertebrates contain surfactant, a complex mixture oflipids and protein that causes surface tension to rise during lunginflation and decrease during lung deflation. During lung deflation,surfactant decreases such that there are no surface forces that wouldotherwise promote alveolar collapse. Aerated alveoli that have notcollapsed during expiration permit continuous oxygen and carbon dioxidetransport between blood and alveolar gas and require much less force toinflate during the subsequent inspiration. During inflation, lungsurfactant increases surface tension as the alveolar surface areaincreases. A rising surface tension in expanding alveoli opposesover-inflation in those airspaces and tends to divert inspired air toless well-aerated alveoli, thereby facilitating even lung aeration.

Respiratory distress syndrome is particularly prevalent among prematureinfants. Lung surfactant is normally synthesized at a very low rateuntil the last six weeks of fetal life. Human infants born more than sixweeks before the normal term of a pregnancy have a high risk of beingborn with inadequate amounts of lung surfactant and inadequate rates ofsurfactant synthesis. The more prematurely an infant is born, the moresevere the surfactant deficiency is likely to be. Severe surfactantdeficiency can lead to respiratory failure within a few minutes or hoursof birth. The surfactant deficiency produces progressive collapse ofalveoli (atelectasis) because of the decreasing ability of the lung toexpand despite maximum inspiratory effort. As a result, inadequateamounts of oxygen reach the infant's blood. RDS can occur in adults aswell, typically as a consequence of failure in surfactant biosynthesis.

Lung tissue of premature infants shows high activity of the hedgehogsignaling pathway. Inhibition of this pathway using hedgehog antagonistsincreases the formation of lamellar bodies and increases the expressionof genes involved in surfactant biosynthesis. Lamellar bodies aresubcellular structures associated with surfactant biosynthesis. Forthese reasons, treatment of premature infants with a hedgehog antagonistshould stimulate surfactant biosynthesis and ameliorate RDS. In caseswhere adult RDS is associated with hedgehog pathway activation,treatment with hedgehog antagonists should also be effective.

It is further contemplated that the use of hedgehog antagonists may bespecifically targeted to disorders where the affected tissue and/orcells evince high hedgehog pathway activation. Expression of gli genesis activated by the hedgehog signaling pathway, including gli-1, gli-2and gli-3. gli-1 expression is most consistently correlated withhedgehog signaling activity across a wide range of tissues anddisorders, while gli-3 is somewhat less so. The gli genes encodetranscription factors that activate expression of many genes needed toelicit the full effects of hedgehog signaling. However, the Gli-3transcription factor can also act as a repressor of hedgehog effectorgenes, and therefore, expression of gli-3 can cause a decreased effectof the hedgehog signaling pathway. Whether Gli-3 acts as atranscriptional activator or repressor depends on post-translationalevents, and therefore it is expected that methods for detecting theactivating form (versus the repressing form) of Gli-3 protein would alsobe a reliable measure of hedgehog pathway activation. gli-2 geneexpression is expected to provide a reliable marker for hedgehog pathwayactivation. The gli-1 gene is strongly expressed in a wide array ofcancers, hyperplasias and immature lungs, and serves as a marker for therelative activation of the hedgehog pathway. In addition, tissues, suchas immature lung, that have high gli gene expression are stronglyaffected by hedgehog inhibitors. Accordingly, it is contemplated thatthe detection of gli gene expression may be used as a powerfulpredictive tool to identify tissues and disorders that will particularlybenefit from treatment with a hedgehog antagonist.

In preferred embodiments, gli-1 expression levels are detected, eitherby direct detection of the transcript or by detection of protein levelsor activity. Transcripts may be detected using any of a wide range oftechniques that depend primarily on hybridization of probes to the gli-1transcripts or to cDNAs synthesized therefrom. Well known techniquesinclude Northern blotting, reverse-transcriptase PCR and microarrayanalysis of transcript levels. Methods for detecting Gli protein levelsinclude Western blotting, immunoprecipitation, two-dimensionalpolyacrylamide gel electrophoresis (2D SDS-PAGE)(preferably comparedagainst a standard wherein the position of the Gli proteins has beendetermined), and mass spectroscopy. Mass spectroscopy may be coupledwith a series of purification steps to allow high-throughputidentification of many different protein levels in a particular sample.Mass spectroscopy and 2D SDS-PAGE can also be used to identifypost-transcriptional modifications to proteins including proteolyticevents, ubiquitination, phosphorylation, lipid modification etc. Gliactivity may also be assessed by analyzing binding to substrate DNA orin vitro transcriptional activation of target promoters. Gel shiftassays, DNA footprinting assays and DNA-protein crosslinking assays areall methods that may be used to assess the presence of a protein capableof binding to Gli binding sites on DNA.

In preferred embodiments, gli transcript levels are measured anddiseased or disordered tissues showing abnormally high gli levels aretreated with a hedgehog antagonist. Premature lung tissue, lung cancers(e.g., adenocarcinomas, broncho-alveolar adenocarcinomas, small cellcarcinomas), breast cancers (e.g., inferior ductal carcinomas, inferiorlobular carcinomas, tubular carcinomas), prostate cancers (e.g.,adenocarcinomas), and benign prostatic hyperplasias all show stronglyelevated gli-1 expression levels in certain cases. Accordingly, gli-1expression levels are a powerful diagnostic device to determine which ofthese tissues should be treated with a hedgehog antagonist. In addition,there is substantial correlative evidence that cancers of urothelialcells (e.g., bladder cancer, other urogenital cancers) will also haveelevated gli-1 levels in certain cases. For example, it is known thatloss of heterozygosity on chromosome 9q22 is common in bladder cancers.The ptc-1 gene is located at this position and ptc-1 loss of function isprobably a partial cause of hyperproliferation, as in many other cancertypes. Accordingly, such cancers would also show high gli expression andwould be particularly amenable to treatment with a hedgehog antagonist.

Expression of ptc-1 and ptc-2 is also activated by the hedgehogsignaling pathway, but these genes are inferior to the gli genes asmarkers of hedgehog pathway activation. In certain tissues only one ofptc-1 or pte-2 is expressed although the hedgehog pathway is highlyactive. For example, in testicular development, Indian hedgehog plays animportant role and the hedgehog pathway is activated, but only ptc-2 isexpressed. Accordingly, these genes are individually unreliable asmarkers for hedgehog pathway activation, although simultaneousmeasurement of both genes are contemplated as a useful indicator fortissues to be treated with a hedgehog antagonist.

Ailments which may be treated by the subject method are disordersspecific to non-humans, such as mange.

In still another embodiment, the subject method can be used in thetreatment of human cancers, particularly basal cell carcinomas and othertumors of epithelial tissues such as the skin. For example, hedgehogantagonists can be employed, in the subject method, as part of atreatment for basal cell nevus syndrome (BCNS), and other humancarcinomas, adenocarcinomas, sarcomas and the like.

Compounds of the invention may be administered prior to, concomitantlywith, or following administration of other anticancer treatments such asradiation therapy or chemotherapy. Suitable cytostatic chemotherapycompounds include, but are not limited to (i) antimetabolites, such ascytarabine, fludarabine, 5-fluoro-2′-deoxyuiridine, gemcitabine,hydroxyurea or methotrexate; (ii) DNA-fragmenting agents, such asbleomycin, (iii) DNA-crosslinking agents, such as chlorambucil,cisplatin, cyclophosphamide or nitrogen mustard; (iv) intercalatingagents such as adriamycin (doxorubicin) or mitoxantrone; (v) proteinsynthesis inhibitors, such as L-asparaginase, cycloheximide, puromycinor diphtheria toxin; (Vi) topoisomerase I poisons, such as camptothecinor topotecan; (vii) topoisomerase II poisons, such as etoposide (VP-16)or teniposide; (viii) microtubule-directed agents, such as colcemid,colchicine, paclitaxel, vinblastine or vincristine; (ix) kinaseinhibitors such as flavopiridol, staurosporine, STI571 (CPG 57148B) orUCN-01 (7-hydroxystaurosporine); (x) miscellaneous investigationalagents such as thioplatin, PS-341, phenylbutyrate, ET-18-OCH₃, orfarnesyl transferase inhibitors (L-739749, L-744832); polyphenols suchas quercetin, resveratrol, piceatannol, epigallocatechine gallate,theaflavins, flavanols, procyanidins, betulinic acid and derivativesthereof; (xi) hormones such as glucocorticoids or fenretinide; (xii)hormone antagonists, such as tamoxifen, finasteride or LHRH antagonists.In a particular embodiment, compounds of the present invention arecoadministered with a cytostatic compound selected from cisplatin,doxorubicin, taxol, taxotere and mitomycin C.

Another class of active compounds which can be used in the presentinvention are those which are able to sensitize for or induce apoptosisby binding to death receptors (“death receptor agonists”). Such agonistsof death receptors include death receptor ligands such as tumor necrosisfactor a (TNF-α), tumor necrosis factor β (TNF-β, lymphotoxin-α), LT-β(lymphotoxin-B), TRAIL (Apo2L, DR4 ligand), CD95 (Fas, APO-1) ligand,TRAMP (DR3, Apo-3) ligand, DR6 ligand as well as fragments andderivatives of any of said ligands. In a particular embodiment, thedeath receptor ligand is TNF-α. In another particular embodiment thedeath receptor ligand is Apo2L/TRAIL. Furthermore, death receptorsagonists comprise agonistic antibodies to death receptors such asanti-CD95 antibody, anti-TRAIL-R1 (DR4) antibody, anti-TRAIL-R2 (DR5)antibody, anti-TRAIL-R3 antibody, anti-TRAIL-R4 antibody, anti-DR6antibody, anti-TNF-R1 antibody and anti-TRAMP (DR3) antibody as well asfragments and derivatives of any of said antibodies.

For the purpose of sensitizing cells for apoptosis, the compounds of thepresent invention can also be used in combination with radiationtherapy. The phrase “radiation therapy” refers to the use ofelectromagnetic or particulate radiation in the treatment of neoplasia.Radiation therapy is based on the principle that high-dose radiationdelivered to a target area will result in the death of reproducing cellsin both tumor and normal tissues. The radiation dosage regimen isgenerally defined in terms of radiation absorbed dose (rad), time andfractionation, and must be carefully defined by the oncologist. Theamount of radiation a patient receives will depend on variousconsideration including the location of the tumor in relation to otherorgans of the body, and the extent to which the tumor has spread.Examples of radiotherapeutic agents are provided in, but not limited to,radiation therapy and is known in the art (Hellman, Principles ofRadiation Therapy, Cancer, in Principles I and Practice of Oncology,24875 (Devita et al., 4th ed., vol 1, 1993). Recent advances inradiation therapy include three-dimensional conformal external beamradiation, intensity modulated radiation therapy (IMRT), stereotacticradiosurgery and brachytherapy (interstitial radiation therapy), thelatter placing the source of radiation directly into the tumor asimplanted “seeds”. These newer treatment modalities deliver greaterdoses of radiation to the tumor, which accounts for their increasedeffectiveness when compared to standard external beam radiation therapy.

Ionizing radiation with beta-emitting radionuclides is considered themost useful for radiotherapeutic applications because of the moderatelinear energy transfer (LET) of the ionizing particle (electron) and itsintermediate range (typically several millimeters in tissue). Gamma raysdeliver dosage at lower levels over much greater distances. Alphaparticles represent the other extreme; they deliver very high LETdosage, but have an extremely limited range and must, therefore, be inintimate contact with the cells of the tissue to be treated. Inaddition, alpha emitters are generally heavy metals, which limits thepossible chemistry and presents hazards from leakage of radionuclidefrom the area to be treated. Depending on the tumor to be treated, allkinds of emitters are conceivable within the scope of the presentinvention. Furthermore, the present invention encompasses types ofnon-ionizing radiation, such as ultraviolet (UV) radiation, high energyvisible light, microwave radiation (hyperthermia therapy), infrared (IR)radiation and lasers. In a particular embodiment of the presentinvention, UV radiation is applied.

In a preferred embodiment, the subject method is used as part of atreatment of prophylaxis regimen for treating (or preventing) basal cellcarcinoma. The deregulation of the hedgehog signaling pathway may be ageneral feature of basal cell carcinomas caused by ptc mutations.Consistent overexpression of human ptc mRNA has been described in tumorsof familial and sporadic BCCs, determined by in situ hybridization.Mutations that inactivate ptc may be expected to result inoverexpression of mutant Ptc, because ptc displays negativeautoregulation. Prior research demonstrates that overexpression ofhedgehog proteins can also lead to tumorigenesis. That sonic hedgehog(Shh) has a role in tumorigenesis in the mouse has been suggested byresearch in which transgenic mice overexpressing Shh in the skindeveloped features of BCNS, including multiple BCC-like epidermalproliferations over the entire skin surface, after only a few days ofskin development. A mutation in the Shh human gene from a BCC was alsodescribed; it was suggested that Shh or other Hh genes in humans couldact as dominant oncogenes in humans. Sporadic ptc mutations have alsobeen observed in BCCs from otherwise normal individuals, some of whichare UV-signature mutations. In one recent study of sporadic BCCs, fiveUV-signature type mutations, either CT or CCTT changes, were found outof fifteen tumors determined to contain ptc mutations. Another recentanalysis of sporadic ptc mutations in BCCs and neuroectodermal tumorsrevealed one CT change in one of three ptc mutations found in the BCCs.See, for example, Goodrich et al. (1997) Science 277:1109-13; Xie et al.(1997) Cancer Res 57:2369-72; Oro et al. (1997) Science 276:817-21; Xieet al. (1997) Genes Chromosomes Cancer 18:305-9; Stone et al. (1996)Nature 384:129-34; and Johnson et al. (1996) Science 272:1668-71.

The subject method can also be used to treatment patients with BCNS,e.g., to prevent BCC or other effects of the disease which may be theresult of ptc loss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function. Basal cell nevus syndrome is a rare autosomal dominantdisorder characterized by multiple BCCs that appear at a young age. BCNSpatients are very susceptible to the development of these tumors; in thesecond decade of life, large numbers appear, mainly on sun-exposed areasof the skin. This disease also causes a number of developmentalabnormalities, including rib, head and face alterations, and sometimespolydactyl), syndactyl), and spina bifida. They also develop a number oftumor types in addition to BCCs: fibromas of the ovaries and heart,cysts of the skin and jaws, and in the central nervous system,medulloblastomas and meningiomas. The subject method can be used toprevent or treat such tumor types in BCNS and non-BCNS patients. Studiesof BCNS patients show that they have both genomic and sporadic mutationsin the ptc gene, suggesting that these mutations are the ultimate causeof this disease.

In another aspect, the present invention provides pharmaceuticalpreparations comprising hedgehog antagonists. The hedgehog antagonistsfor use in the subject method may be conveniently formulated foradministration with a biologically acceptable medium, such as water,buffered saline, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol and the like) or suitable mixtures thereof. Theoptimum concentration of the active ingredient(s) in the chosen mediumcan be determined empirically, according to procedures well known tomecLicinal chemists. As used herein, “biologically acceptable medium”includes any and all solvents, dispersion media, and the like which maybe appropriate for the desired route of administration of thepharmaceutical preparation. The use of such media for pharmaceuticallyactive substances is known in the art. Except insofar as anyconventional media or agent is incompatible with the activity of thehedgehog antagonist, its use in the pharmaceutical preparation of theinvention is contemplated. Suitable vehicles and their formulationinclusive of other proteins are described, for example, in the bookRemington's Pharmaceutical Sciences (Remington's PharmaceuticalSciences. Mack Publishing Company, Easton, Pa., USA 1985). Thesevehicles include injectable “deposit formulations”.

Pharmaceutical formulations of the present invention can also includeveterinary compositions, e.g., pharmaceutical preparations of thehedgehog antagonists suitable for veterinary uses, e.g., for thetreatment of livestock or domestic animals, e.g., dogs.

Methods of introduction may also be provided by rechargeable orbiodegradable devices. Various slow release polymeric devices have beendeveloped and tested in vivo in recent years for the controlled deliveryof drugs, including proteinacious biopharmaceuticals. A variety ofbiocompatible polymers (including hydrogels), including bothbiodegradable and non-degradable polymers, can be used to form animplant for the sustained release of a hedgehog antagonist at aparticular target site.

The composition of the invention will be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. The“effective amount” of the compound to be administered will be governedby such considerations, and is the minimum amount necessary to decreasehedgehog pathway signaling or else is the minimum amount necessary tocause reduction in size, volume or mass of a tumor that is responsive tohedgehog signaling, or a reduction in the increase in size, volume ormass of such a tumor relative to the increase in the absence ofadministering the compound of the invention. Alternatively “effectiveamount” of the compound means the amount necessary to reduce the numberof malignant cells or the rate in increase of the number of malignantcells. Alternatively, “effective amount” is the amount of the compoundof the invention required to increase survival of patients afflictedwith an anti-hedgehog pathway sensitive tumor. Such amount may be belowthe amount that is toxic to normal cells, or the mammal as a whole. Withrespect to non-malignant indications, “effective amount” means theamount of compound of the invention required to decrease severity of theparticular indication or symptoms thereof.

Generally, the initial pharmaceutically effective amount of the compoundof the invention administered parenterally per dose will be in the rangeof about 0.01 to about 100 mg/kg, for example about 0.1 to about 20mg/kg of patient body weight per day, for example about 0.3 to about 15mg/kg/day. Oral unit dosage forms, such as tablets and capsules, maycontain from about 25 to about 1000 mg of the compound of the invention.

The preparations of the invention may be given by any suitable means,including oral, topical, transdermal, parenteral, subcutaneous, rectal,intraperitoneal, intrapulmonary, and intranasal, and, if desired forlocal treatment, intralesional administration. They are of course givenby forms suitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, controlled release patch, etc.administration by injection, infusion or inhalation; topical by lotionor ointment; and rectal by suppositories. Oral and topicaladministrations are preferred. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. An example of a suitable oral dosage formis a tablet containing about 25 mg, 50 mg, 100 mg, 250 mg, or 500 mg ofthe compound of the invention compounded with about 90-30 mg anhydrouslactose, about 5-40 mg sodium croscarmellose, about 5-30 mgpolyvinylpyrrolidone (PVP) K30, and about 1-10 mg magnesium stearate.The powdered ingredients are first mixed together and then mixed with asolution of the PVP. The resulting composition can be dried, granulated,mixed with the magnesium stearate and compressed to tablet form usingconventional equipment. An aerosol formulation can be prepared bydissolving the compound, for example, 5-400 mg, of the invention in asuitable buffer solution, e.g., a phosphate buffer, adding a tonicifier,e.g., a salt such as sodium chloride, if desired. The solution istypically filtered, e.g., using a 0.2 micron filter, to removeimpurities and contaminants. Topical formulations include ointments,creams, lotions, powders, solutions, pessaries, sprays, aerosols andcapsules. Ointments and creams may be formulated with an aqueous or oilybase with the addition of suitable thickening and/or gelling agentsand/or solvents. Such bases may include water and/or an oil such aliquid paraffin or a vegetable oil such as arachis oil or castor oil ora solvent such as a polyethylene glycol. Thickening agents which may beused include soft paraffin, aluminum stearate, cetostearyl alcohol,polyethylene glycols, microcrystalline wax and beeswax. Lotions may beformulated with an aqueous or oily base and may contain one or moreemulsifying agents, stabilizing agents, dispersing agents, suspendingagents or thickening agents. Powders for external application may beformed with the aid of any suitable powder base, e.g., talc, lactose orstarch. Drops may be formulated with an aqueous or non-aqueous base alsocomprising one or more dispersing agents, solubilizing agents orsuspending agents.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms such as described below orby other conventional methods known to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular hedgehog antagonist employed, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound that is the lowest dose effective to producea therapeutic effect. Such an effective dose will generally depend uponthe factors described above. Generally, intravenous,intracerebroventricular and subcutaneous doses of the compounds of thisinvention for a patient will range from about 0.0001 to about 100 mg perkilogram of body weight per day.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

The compound of the invention can be administered as such or inadmixtures with pharmaceutically acceptable and/or sterile carriers andcan also be administered in conjunction with other antimicrobial agentssuch as penicillins, cephalosporins, aminoglycosides and glycopeptides.Conjunctive therapy, thus includes sequential, simultaneous and separateadministration of the active compound in a way that the therapeuticaleffects of the first administered one is not entirely disappeared whenthe subsequent is administered.

V. Pharmaceutical Compositions

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical formulation (composition). The hedgehog antagonistsaccording to the invention may be formulated for administration in anyconvenient way for use in human or veterinary medicine. In certainembodiments, the compound included in the pharmaceutical preparation maybe active itself, or may be a prodrug, e.g., capable of being convertedto an active compound in a physiological setting.

Thus, another aspect of the present invention provides pharmaceuticallyacceptable compositions comprising a therapeutically effective amount ofone or more of the compounds described above, formulated together withone or more pharmaceutically acceptable carriers (additives) and/ordiluents. As described in detail below, the pharmaceutical compositionsof the present invention may be specially formulated for administrationin solid or liquid form, including those adapted for the following: (1)oral administration, for example, drenches (aqueous or non-aqueoussolutions or suspensions), tablets, boluses, powders, granules, pastesfor application to the tongue; (2) parenteral administration, forexample, by subcutaneous, intramuscular or intravenous injection as, forexample, a sterile solution or suspension; (3) topical application, forexample, as a cream, ointment or spray applied to the skin; or (4)intravaginally or intrarectally, for example, as a pessary, cream orfoam. However, in certain embodiments the subject compounds may besimply dissolved or suspended in sterile water. In certain embodiments,the pharmaceutical preparation is non-pyrogenic, i.e., does not elevatethe body temperature of a patient.

The phrase “therapeutically effective amount” as used herein means thatamount of a compound, material, or composition comprising a compound ofthe present invention which is effective for producing some desiredtherapeutic effect by overcoming a ptc loss-of-function, hedgehoggain-of-function, or smoothened gain-of-function in at least asub-population of cells in an animal and thereby blocking the biologicalconsequences of that pathway in the treated cells, at a reasonablebenefit/risk ratio applicable to any medical treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject antagonistsfrom one organ, or portion of the body, to another organ, or portion ofthe body. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

As set out above, certain embodiments of the present hedgehogantagonists may contain a basic functional group, such as amino oralkylamino, and are, thus, capable of forming pharmaceuticallyacceptable salts with pharmaceutically acceptable acids. The term“pharmaceutically acceptable salts” in this respect, refers to therelatively non-toxic, inorganic and organic acid addition salts ofcompounds of the present invention. These salts can be prepared in situduring the final isolation and purification of the compounds of theinvention, or by separately reacting a purified compound of theinvention in its free base form with a suitable organic or inorganicacid, and isolating the salt thus formed. Representative salts includethe hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate,acetate, valerate, oleate, palmitate, stearate, laurate, benzoate,lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate,tartrate, napthylate, mesylate, glucoheptonate, lactobionate, andlaurylsulphonate salts and the like. (See, for example, Berge et al.(1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19)

The pharmaceutically acceptable salts of the subject compounds includethe conventional nontoxic salts or quaternary ammonium salts of thecompounds, e.g., from non-toxic organic or inorganic acids. For example,such conventional nontoxic salts include those derived from inorganicacids such as hydrochloride, hydrobromic, sulfuric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically acceptable salts with pharmaceutically acceptablebases. The term “pharmaceutically acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can likewise beprepared in situ during the final isolation and purification of thecompounds, or by separately reacting the purified compound in its freeacid form with a suitable base, such as the hydroxide, carbonate OTbicarbonate of a pharmaceutically acceptable metal cation, with ammonia,or with a pharmaceutically acceptable organic primary, secondary ortertiary amine. Representative alkali or alkaline earth salts includethe lithium, sodium, potassium, calcium, magnesium, and aluminum saltsand the like. Representative organic amines useful for the formation ofbase addition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine and the like. (See, forexample, Berge et al., supra)

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient that can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.Generally, out of one hundred percent, this amount will range from about1 percent to about ninety-nine percent of active ingredient, preferablyfrom about 5 percent to about 70 percent, most preferably from about 10percent to about 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols andthe like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions that can bedissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions that can be used include polymeric substances andwaxes. The active ingredient can also be in micro-encapsulated form, ifappropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

It is known that sterols, such as cholesterol, will form complexes withcyclodextrins. Thus, in preferred embodiments, where the inhibitor is asteroidal alkaloid, it may be formulated with cyclodextrins, such as α-,β- and γ-cyclodextrin, dimethyl-β cyclodextrin and2-hydroxypropyl-β-cyclodextrin.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active hedgehog antagonist.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants that may berequired.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the hedgehog antagonistsin the proper medium. Absorption enhancers can also be used to increasethe flux of the hedgehog antagonists across the skin. The rate of suchflux can be controlled by either providing a rate controlling membraneor dispersing the compound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents that delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide.

Depending on the ratio of drug to polymer, and the nature of theparticular polymer employed, the rate of drug release can be controlled.Examples of other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions that are compatiblewith body tissue.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99.5% (morepreferably, 0.5 to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

The addition of the active compound of the invention to animal feed ispreferably accomplished by preparing an appropriate feed premixcontaining the active compound in an effective amount and incorporatingthe premix into the complete ration.

Alternatively, an intermediate concentrate or feed supplement containingthe active ingredient can be blended into the feed. The way in whichsuch feed premixes and complete rations can be prepared and administeredare described in reference books (such as “Applied Animal Nutrition”,W.H. Freedman and CO., San Francisco, U.S.A., 1969 or “Livestock Feedsand Feeding” 0 and B books, Corvallis, Ore., U.S.A., 1977).

VI. Synthetic Preparation and Identification of Active Antagonists

The subjects steroidal alkaloids, and congeners thereof, can be preparedreadily by employing the cross-coupling technologies of Suzuki, Stille,and the like. These coupling reactions are carried out under relativelymild conditions and tolerate a wide range of “spectator” functionality.

a. Combinatorial Libraries

The compounds of the present invention, particularly libraries ofvariants having various representative classes of substituents, areamenable to combinatorial chemistry and other parallel synthesis schemes(see, for example, PCT WO 94/08051). The result is that large librariesof related compounds, e.g. a variegated library of compounds representedabove, can be screened rapidly in high throughput assays in order toidentify potential hedgehog antagonist lead compounds, as well as torefine the specificity, toxicity, and/or cytotoxic-kinetic profile of alead compound. For instance, ptc, hedgehog, or smoothened bioactivityassays, such as may be developed using cells with either a ptcloss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function, can be used to screen a library of the subjectcompounds for those having agonist activity toward ptc or antagonistactivity towards hedgehog or smoothened.

Simply for illustration, a combinatorial library for the purposes of thepresent invention is a mixture of chemically related compounds that maybe screened together for a desired property. The preparation of manyrelated compounds in a single reaction greatly reduces and simplifiesthe number of screening processes that need to be carried out. Screeningfor the appropriate physical properties can be done by conventionalmethods.

Diversity in the library can be created at a variety of differentlevels. For instance, the substrate aryl groups used in thecombinatorial reactions can be diverse in terms of the core aryl moiety,e.g., a variegation in terms of the ring structure, and/or can be variedwith respect to the other substituents.

A variety of techniques are available in the art for generatingcombinatorial libraries of small organic molecules such as the subjecthedgehog antagonists. See, for example, Blondelle et al. (1995) TrendsAnal. Chem. 14:83; the Affymax U.S. Pat. Nos. 5,359,115 and 5,362,899:the Ellman U.S. Pat. No. 5,288,514: the Still et al. PCT publication WO94/08051; the ArQule U.S. Pat. Nos. 5,736,412 and 5,712,171; Chen et al.(1994) JACS 116:2661: Kerr et al. (1993) JACS 115:252; PCT publicationsWO92/10092, WO93/09668 and WO91/07087; and the Lerner et al. PCTpublication WO93/20242). Accordingly, a variety of libraries on theorder of about 100 to 1,000,000 or more diversomers of the subjecthedgehog antagonists can be synthesized and screened for particularactivity or property.

In an exemplary embodiment, a library of candidate hedgehog antagonistsdiversomers can be synthesized utilizing a scheme adapted to thetechniques described in the Still et al. PCT publication WO 94/08051,e.g., being linked to a polymer bead by a hydrolyzable or photolyzablegroup, optionally located at one of the positions of the candidateantagonists or a substituent of a synthetic intermediate. According tothe Still et al. technique, the library is synthesized on a set ofbeads, each bead including a set of tags identifying the particulardiversomer on that bead. The bead library can then be “plated” with ptcloss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function cells for which an hedgehog antagonist is sought. Thediversomers can be released from the bead, e.g. by hydrolysis.

The structures of the compounds useful in the present invention lendthemselves readily to efficient synthesis. The nature of the structures,as generally described by formulas I to II, allows the combinatorialassembly of such compounds using some combination of Ar, R₁, X, Y, and Zmoieties, as set forth above. For example, these subunits can beattached to the core ring through common acylation or alkylationreactions. The vast majority of such reactions are both extremely mildand extremely reliable, and are thus perfectly suited for combinatorialchemistry. Such combinatorial approaches may employ variations on one ofthe routes disclosed below.

Many variations on the above and related pathways permit the synthesisof widely diverse libraries of compounds that may be tested asinhibitors of hedgehog function.

b. Screening Assays

There are a variety of assays available for determining the ability of acompound to agonize ptc function or antagonize smoothened or hedgehogfunction, many of which can be disposed in high-throughput formats. Inmany drug-screening programs that test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Thus, libraries of synthetic and natural products can be sampled forother compounds that are hedgehog antagonists.

In addition to cell-free assays, test compounds can also be tested incell-based assays. In one embodiment, cell which have a ptcloss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function phenotype can be contacted with a test agent ofinterest, with the assay scoring for, e.g., inhibition of proliferationof the cell in the presence of the test agent.

A number of gene products have been implicated in patched-mediatedsignal transduction, including patched, transcription factors of thecubitus interruptus (ci) family, the serine/threonine kinase fused (fu)and the gene products of costal-2, smoothened and suppressor of fused.

The induction of cells by hedgehog proteins sets in motion a cascadeinvolving the activation and inhibition of downstream effectors, theultimate consequence of which is, in some instances, a detectable changein the transcription or translation of a gene. Potential transcriptionaltargets of hedgehog-mediated signaling are the patched gene (Hidalgo andIngham, 1990 Development 110, 291-301; Marigo et al., 1996) and thevertebrate homologs of the drosophila cubitus interruptus gene, the GLIgenes (Hui et al. (1994) Dev Biol 162:402-413). Patched gene expressionhas been shown to be induced in cells of the limb bud and the neuralplate that are responsive to Shh. (Marigo et al. (1996) PNAS 93:9346-51;Marigo et al. (1996) Development 122:1225-1233). The Gli genes encodeputative transcription factors having zinc finger DNA binding domains(Orenic et al. (1990) Genes & Dev 4:1053-1067; Kinzler et al. (1990) MolCell Biol 10:634-642). Transcription of the Gli gene has been reportedto be upregulated in response to hedgehog in limb buds, whiletranscription of the Gli3 gene is downregulated in response to hedgehoginduction (Marigo et al. (1996) Development 122:1225-1233). By selectingtranscriptional regulatory sequences from such target genes, e.g., frompatched or Gli genes, that are responsible for the up- ordown-regulation of these genes in response to hedgehog signalling, andoperatively linking such promoters to a reporter gene, one can derive atranscription based assay which is sensitive to the ability of aspecific test compound to modify hedgehog-mediated signalling pathways.Expression of the reporter gene, thus, provides a valuable screeningtool for the development of compounds that act as antagonists ofhedgehog.

Reporter gene based assays of this invention measure the end stage ofthe above described cascade of events, e.g., transcriptional modulation.Accordingly, in practicing one embodiment of the assay, a reporter geneconstruct is inserted into the reagent cell in order to generate adetection signal dependent on ptc loss-of-function, hedgehoggain-of-function, smoothened gain-of-function, or stimulation by SHHitself. The amount of transcription from the reporter gene may bemeasured using any method known to those of skill in the art to besuitable. For example, mRNA expression from the reporter gene may bedetected using RNAse protection or RNA-based PCR, or the protein productof the reporter gene may be identified by a characteristic stain or anintrinsic biological activity. The amount of expression from thereporter gene is then compared to the amount of expression in either thesame cell in the absence of the test compound or it may be compared withthe amount of transcription in a substantially identical cell that lacksthe target receptor protein. Any statistically or otherwise significantdecrease in the amount of transcription indicates that the test compoundhas in some manner agonized the normal ptc signal (or antagonized thegain-of-function hedgehog or smoothened signal), e.g., the test compoundis a potential hedgehog antagonist.

c. Exemplified Compounds

Table 1 contains some of the compounds that exemplify Formulas I, II,II, and IV. The examples in Table 1 are in no way meant to be limiting.

TABLE 1 Exam- ple Compound 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

VII. Business Methods

One aspect of the present invention relates to a kit comprising ahedgehog antagonist, e.g., as described herein, for treating orpreventing basal cell carcinoma in a patient, preferably a human, and inassociation with instructions (written and/or pictorial) describing theuse of the formulation for treatment or prevention of basal cellcarcinoma, and, optionally, warnings of possible side effects anddrug-drug or drug-food interactions.

Another aspect of the present invention relates to a kit comprising ahedgehog antagonist, e.g., as described herein, wherein the hedgehogantagonist is administered as part of a therapeutic or cosmeticapplication, such as those described above (e.g., regulation of neuraltissues, bone and cartilage formation and repair, regulation ofspermatogenesis, regulation of smooth muscle, regulation of lung, liverand other organs arising from the primative gut, regulation ofhematopoietic function, and regulation of skin and hair growth), to apatient, preferably a human, in association with instructions (writtenand/or pictorial) describing the therapeutic or cosmetic application,and optionally, warnings of possible side effects and drug-drug ordrug-food interactions.

The invention further contemplates a method for conducting apharmaceutical business, comprising: (a) manufacturing a pharmaceuticalpreparation comprising a sterile pharmaceutical excipient and a hedgehogantagonist; and (b) marketing (e.g., providing promotional and/or informactive presentations (such as displays, telemarketing, and lectures),products (such as trial samples of the preparation), and/ordocumentation (including leaflets, pamphlets, websites, posters, etc.))to healthcare providers, such as doctors, hospitals, clinics, etc., abenefit of using the pharmaceutical preparation for treating orpreventing basal cell carcinoma.

Another aspect of the present invention relates to a method forconducting a pharmaceutical business, comprising: (a) manufacturing apharmaceutical preparation comprising a sterile pharmaceutical excipientand a hedgehog antagonist; and (b) marketing (e.g., providingpromotional and/or informative presentations (such as displays,demonstrations, telemarketing, and lectures), products (such as trialsamples of the preparation), and/or documentation (including leaflets,pamphlets, websites, posters, etc.)) to healthcare providers, such asdoctors, hospitals, clinics, etc., a benefit of using the pharmaceuticalpreparation as part of a therapeutic or cosmetic application, such asregulation of neural tissues, bone and cartilage formation and repair,regulation of spermatogenesis, regulation of smooth muscle, regulationof lung, liver and other organs arising from the primitive gut,regulation of hematopoietic function, and regulation of skin and hairgrowth.

Another aspect of the invention provides for a method for conducting apharmaceutical business, comprising: (a) providing a distributionnetwork for selling the pharmaceutical composition comprising a sterilepharmaceutical excipient and a hedgehog antagonist; and (b) providinginstruction material to patients or physicians for using thepharmaceutical composition for treating or preventing basal cellcarcinoma.

Another aspect of the invention provides for a method for conducting apharmaceutical business, comprising: (a) providing a distributionnetwork for selling the pharmaceutical composition comprising a sterilepharmaceutical excipient and a hedgehog antagonist; and (b) providinginstruction material to patients or physicians for using thepharmaceutical composition as part of a therapeutic or cosmeticapplication, such as those described above (e.g., regulation of neuraltissues, bone and cartilage formation and repair, regulation ofspermatogenesis, regulation of smooth muscle, regulation of lung, liverand other organs arising from the primative gut, regulation ofhematopoietic function, and regulation of skin and hair growth).

Another aspect of the invention provides for a method for conducting apharmaceutical business, comprising: (a) determining an appropriatepharmaceutical preparation and dosage of a hedgehog antagonist fortreatment or prevention of basal cell carcinoma; (b) conductingtherapeutic profiling of the pharmaceutical composition for efficacy andtoxicity in animals; (c) providing a distribution network for selling apharmaceutical composition having an acceptable therapeutic profile;and, optionally, (d) providing a sales group for marketing thepreparation to healthcare providers.

Another aspect of the invention provides for a method for conducting apharmaceutical business, comprising: (a) determining an appropriatepharmaceutical preparation and dosage of a hedgehog antagonist fortreatment or prevention of a medical or cosmetic condition describedabove; (b) conducting therapeutic profiling of the pharmaceuticalcomposition for efficacy and toxicity in animals in treating orpreventing the condition; (c) providing a distribution network forselling a pharmaceutical composition having an acceptable therapeuticprofile for treating or preventing the condition; and, optionally, (d)providing a sales group for marketing the preparation to healthcareproviders for the treatment or prevention of the condition.

Yet another aspect of the invention provides a method for conducting apharmaceutical business, comprising: (a) determining an appropriatepharmaceutical composition and dosage of a hedgehog antagonist fortreatment or prevention of basal cell carcinoma; and (b) licensing, to athird party, rights for further development and sale of thepharmaceutical composition.

Yet another aspect of the invention provides a method for conducting apharmaceutical business, comprising: (a) determining an appropriatepharmaceutical composition and dosage of a hedgehog antagonist fortreatment or prevention of a medical or cosmetic condition as describedabove; and (b) licensing, to a third party, rights for furtherdevelopment and sale of the pharmaceutical composition for the treatmentor prevention of the condition.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

EXPERIMENTAL Synthesis of Exemplary Inhibitors

Exemplified for compound 24

To a solution of 2-methyl-6-trifluoromethylnicotinic acid (4.77 g; 23mmol) in dimethyl formamide (100 ml) was added1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.46 g; 23mmol) and 1-hydroxy-7-azabenzotriazole (3.17 g; 23 mmol) and theresulting solution was stirred at room temperature for 30 minutes. Tothis was added a solution of methyl 5-amino-2-chlorobenzoate (3.6 g; 19mmol) in dimethyl formamide (25 ml) and the resulting solution stirredat room temperature for 18 hours. Water (200 ml) was added to thereaction mixture and this was extracted with ethyl acetate (3×200 ml).The combined organic layers were washed with saturated sodiumbicarbonate solution (2×300 ml). The organic layer was dried (magnesiumsulphate), filtered and evaporated to give2-chloro-5-[(2-methyl-6-trifluoromethyl-pyridine-3-carbonyl)-amino]-benzoicacid methyl ester (6.6 g, 94%).

A solution of2-chloro-5-[(2-methyl-6-trifluoromethyl-pyridine-3-carbonyl)-amino]-benzoicacid methyl ester (6.68 g; 18 mmol) in tetrahydrofuran (100 ml) wascooled to 0° C. Lithium borohydride (0.98 g; 45 mmol) was addedportionwise and the reaction mixture allowed to warn to roomtemperature, followed by heating at 45° C. for 4 hours. The reaction wasallowed to cool to room temperature and saturated sodium bicarbonatesolution (100 ml) was added. This was extracted with ethyl acetate (3×50ml). The organic layers were dried (magnesium sulphate), filtered andevaporated. The residue was purified by column chromatography (50% ethylacetate in heptane) to giveN-(4-chloro-3-hydroxymethyl-phenyl)-2-methyl-6-trifluoromethyl-nicotinamide(3.29 g, 54%).

To a suspension ofN-(4-chloro-3-hydroxymethyl-phenyl)-2-methyl-6-trifluoromethyl-nicotinamide(3.29 g; 9.56 mmol) in dichloromethane (300 ml) was added manganesedioxide (8.31 g; 95.6 mmol) and the resulting mixture stirred at roomtemperature for 30 minutes. Analysis after this time showed a 60%conversion to product. Further manganese dioxide (8 g; 92 mmol) wasadded in three portions over the next 90 minutes. The reaction mixturewas then filtered through celite and the filtrate evaporated to giveN-(4-chloro-3-formyl-phenyl)-2-methyl-6-trifluoromethyl-nicotinamide(3.2 g, 97%).

To a solution ofN-(4-chloro-3-formyl-phenyl)-2-methyl-6-trifluoromethyl-nicotinamide(152 mg; 0.44 mmol) and benzo[1,3]dioxole-5,6-diamine (100 mg; 0.44mmol) in N-methylpyrrolidone (3 ml) was added triethylamine (0.12 ml;0.88 mmol). The resulting solution was heated to 100° C. for 1 hour.Water (10 ml) was added to the cooled reaction mixture and this wasextracted with ethyl acetate (3×10 ml). The organic layer was dried(magnesium sulphate), filtered and evaporated to give the crude residue.This was purified by column chromatography (30% ethyl acetate inheptane) to give compound 24 (146 mg, 70%): ¹H NMR (400 MHz, MeOD) δ8.45 (s, 1H), 8.0 (d, 1H), 7.65-7.75 (m, 3H), 7.2 (s, 2H), 6.1 (s, 2H),2.65 (s, 3H); MS (ES⁺): 475 (³⁵Cl), 477 (³⁷Cl) [M⁺+1].

Exemplified for compound 5

5-Chloro-2-nitroaniline (1 g; 5.8 mmol) and pyridine (0.64 ml; 8.1 mmol)were dissolved in dichloromethane (10 ml) and cooled to 0° C. This wasadded dropwise over 5 minutes to a solution of 2-chloro-5-nitrobenzoylchloride (1.34 g; 6.1 mmol) in dichloromethane (5 ml). The reaction wasstirred at room temperature for 72 hours before adding further acidchloride (1.34 g) and pyridine (0.64 ml). After 30 minutes, the solventwas evaporated and the bisacylated residue treated with 1:1 concentratedammonia/acetonitrile (10 ml). All the solvent was removed after 5minutes and the residue crystallised from 1:1 chloroform/heptane (30 ml)to give 2-chloro-N-(5-chloro-2-nitro-phenyl)-5-nitro-benzamide (1.78 g,86%)

2-Chloro-N-(5-chloro-2-nitro-phenyl)-5-nitro-benzamide (1.78 g; 5 mmol)was dissolved in glacial acetic acid (30 ml) and heated to 70° C. Zincpowder (3.3 g; 50 mmol) was added portionwise. After 2 hours, thereaction was filtered and the solid washed with acetonitrile. Thesolvents were evaporated and the residue loaded onto a silica gelchromatography column using dichloromethane/triethylamine. Elution with20% ethyl acetate in heptane gave4-chloro-3-(5-chloro-1H-benzoimidazol-2-yl)-phenylamine as an orangepowder (0.22 g, 16%).

4-Chloro-3-(5-chloro-1H-benzoimidazol-2-yl)-phenylamine (80 mg; 0.29mmol) and triethylamine (23 μl; 0.32 mmol) were dissolved intetrahydrofuran (2 ml). A solution of2-methyl-6-trifluoromethylnicotinoyl chloride (58 mg; 0.26 mmol) intetrahydrofuran (1 ml) was added over 5 minutes. After stirring for 6hours at room temperature, the solvent was evaporated and the residueloaded onto a silica gel chromatography using a dichloromethane/methanolmixture. Elution with 10% methanol in dichloromethane gave compound 5 asthe free base. This was dissolved in methanol (1 ml) and concentratedhydrochloric acid (2 drops) added. Evaporation provided thehydrochloride salt (56 mg, 43%): ¹H NMR (400 MHz, d₆ DMSO) δ11.1 (s,1H), 8.45 (s, 1H), 8.25 (d, 1H), 7.9 (m, 2H), 7.85 (s, 1H), 7.75 (m,2H), 7.45 (d, 1H), 2.65 (s, 3H); MS (ES⁺): 465 (³⁵Cl), 467 (³⁷Cl)[M⁺+1].

5-Chloro-2-nitroaniline (50 g; 0.29 mol) was added to a 5 L bombfollowed by ethanol (1.1 L) at 0-5° C. Dimethylamine hydrochloride (130g; 1.59 mmol) was added in one portion followed by triethylamine (260ml) and the bomb quickly sealed. The bomb was heated to 130° C. withstirring at 4 bar for 20 hours. The reaction mixture was then allowed tocool to room temperature and the ethanol was removed under vacuum. 2 Msodium hydroxide solution (1 L) was added to the crude residue and thiswas extracted with ethyl acetate (1×1.3 L then 2×500 ml). The combinedorganic layers were washed with brine (150 ml), dried (magnesiumsulphate), filtered and evaporated to give the5-dimethylamino-2-nitroaniline (50.1 g, 96%).

Compound 2 was prepared from 5-dimethylamino-2-nitroaniline using route2: ¹H NMR (400 MHz, d₆ DMSO) δ 11.35 (brs, 1H), 8.6 (s, 1H), 8.4 (d,1H), 8.1 (m, 3H), 7.95 (d, 1H), 7.6-7.7 (brm, 1H), 3.3 (s, 6H), 2.85 (s,3H); MS (ES⁺): 474 (³⁵Cl), 476 (³⁷Cl) [M⁺+1].

2-Aminophenol (300 mg; 2.75 mmol) and 2-chloro-5-nitrobenzoyl chloride(606 mg; 2.75 mmol) were dissolved in N-methylpyrrolidone (6 ml) andheated at 100° C. for 24 hours. The reaction was cooled to roomtemperature, partitioned between ethyl acetate and saturated sodiumbicarbonate, and the organic layer dried (sodium sulphate). Afterfiltration and evaporation, the product was purified by columnchromatography on silica gel. 2-(2-Chloro-5-nitro-phenyl)-benzooxazoleeluted with 40% ethyl acetate in heptane (320 mg, 42%)

2-(2-Chloro-5-nitro-phenyl)-benzooxazole was reduced to the aniline asdescribed for compound 16 and the aniline converted to compound 3 as inroute 2 step 3. ¹H NMR (400 MHz, d₆ DMSO) δ11.25 (s, 1H), 8.95 (s, 1H),8.55 (d, 1H), 8.1-8.2 (m, 4H), 8.0 (d, 1H), 7.7-7.8 (m, 2H), 2.95 (s,3H); MS (ES⁺): 432 (³⁵Cl), 434 (³⁷Cl) [M⁺+1].

2-Nitro-5-(1-pyrrolo)aniline was prepared using the procedure describedfor compound 11.

Compound 6 was prepared from 2-nitro-5-(1-pyrrolo)aniline using route 2:¹H NMR (400 MHz, MeOD) δ 8.55 (s, 1H), 8.05 (d, 1H), 7.85 (dd, 2H), 7.75(d, 2H), 7.7 (dd, 2H), 7.25 (s, 2H), 6.25 (s, 2H), 2.65 (s, 3H); MS(ES⁺): 496 (³⁵Cl), 498 (³⁷Cl) [M⁺+1].

Fuming white nitric acid (3.6 g) was added dropwise to acetic anhydride(12 ml) at 0° C. and stirred for 15 minutes. TheN-(3-acetyl-phenyl)-acetamide (2 g; 11.3 mmol) was then addedportionwise and the reaction mixture allowed to warm to roomtemperature. After stirring at room temperature for 15 minutes, thereaction mixture was poured onto ice (200 ml). Dichloromethane (200 ml)was added and the organic layer was separated, dried (magnesiumsulphate), filtered and evaporated to give the crude residue. This waspurified by column chromatography to giveN-(5-acetyl-2-nitro-phenyl)-acetamide (515 mg), which was directlyhydrolysed by heating in 6M hydrochloric acid (15 ml) at 80° C. for 2hours. The cooled reaction mixture was basified using saturated sodiumbicarbonate solution and then extracted into ethyl acetate (6×30 ml).The organic layers were dried (magnesium sulphate), filtered andevaporated to give the crude residue. This was purified by columnchromatography (50% ethyl acetate in heptane) to give3-amino-4-nitroacetophenone (226 mg, 11%).

Compound 7 was prepared from 3-amino-4-nitroacetophenone using route 2:¹H NMR (400 MHz, MeOD) δ 8.2 8.3 (brs, 1H), 8.15 (s, 1H), 8.05 (d, 1H),7.95 (d, 1H), 7.85 (d, 1H), 7.7 (d, 1H), 7.65 (brs, 1H), 7.55 (d, 1H),2.65 (s, 3H), 2.60 (s, 3H); MS (ES⁺): 473 (³⁵Cl), 475 (³⁷Cl) [M⁺+1].

In a microwave tube, a solution of 4-amino-2-chloro-5-nitropyridine(prepared as described for compound 9) (200 mg; 1.16 mmol) in ethanol (2ml) was treated sequentially with dimethylamine hydrochloride (471 mg;5.78 mmol) and triethylamine (781 μl; 6.96 mmol). The tube was quicklysealed and irradiated in the microwave (CEM Discover, 150 W, 85° C., 10min). The ethanol was evaporated, dichloromethane (15 ml) was added todissolve the products, and the solution washed with 1M sodium hydroxidesolution (2×10 ml). The organic layer was dried (sodium sulphate), themixture filtered and the filtrate evaporated to dryness to affordN²,N²-dimethyl-5-nitro-pyridine-2,4-diamine (240 mg, 114%).

N²,N²-dimethyl-5-nitro-pyridine-2,4-diamine was converted into compound8 using the method described in route 2: ¹H NMR (400 MHz, MeOD) δ 8.44(s, 1H), 8.13 (s, 1H), 8.00 (d, 1H), 7.78 (d, 1H), 7.64 (d, 1H), 7.52(d, 1H), 6.55 (bs, 1H), 3.04 (s, 6H) and 2.58 (s, 3H); MS (ES⁺): 475(³⁵Cl), 477 (³⁷Cl) [M⁺+1].

To stirred concentrated sulphuric acid (100 ml) at 4° C. was added2-chloro-4-aminopyridine (35.0 g; 0.273 mol) in portions. White fumingnitric acid (26 ml) was added dropwise over 20 minutes and the mixturestirred at 4° C. for 1 hour before being poured onto ice (1 L). Theresultant mixture was taken to pH 2 with 6 M NaOH at which point a thickprecipitate formed. This was filtered, washed with water and dried inair to afford the nitramine intermediate which was used in the next stepwithout further purification.

A solution of the nitramine (42 g) in concentrated sulphuric acid (100ml) was heated at 85° C. for 2 hours, then at 110° C. for a further 1hour. The mixture was cooled, poured into ice (1 L) and basified withconcentrated ammonia solution to pH 9. The resultant suspension wasfiltered and the solid washed with water. The solid was then extractedwith hot toluene (3×250 ml), and the combined extracts evaporated andchromatographed (heptane: ethyl acetate 9:2) to afford2-chloro-4-amino-5-nitropyridine as a white solid (4.57 g, 10%); ¹H NMR(400 MHz, DMSO) δ 8.85 (1H, d), 7.90-8.22 (2H, bs) and 6.90 (1H, d).

4-Amino-2-chloro-5-nitropyridine was converted into compound 9 usingroute 2: ¹H NMR (400 MHz, MeOD) δ 8.64 (s, 1H), 8.21 (s, 1H), 8.03 (d,1H), 7.82 (d, 1H), 7.65 (d, 1H), 7.58-7.56 (m, 2H) and 2.60 (s, 3H); MS(ES⁺): 466 (³⁵Cl, ³⁵Cl), 468 (³⁵Cl, ³⁷C), 470 (³⁷Cl, ³⁷C)[M⁺+1].

Compound 10 was prepared from 2-aminothiophenol using the methoddescribed in route 1 step 4: ¹H NMR (400 MHz, MeOD) δ 8.45 (s, 1H), δ8.05 (m, 3H), 7.8 (d, 1H), 7.65 (d, 1H), 7.5-7.55 (m, 2H), 7.4 (dd, 1H),2.65 (s, 3H); MS (ES⁺): 448 (³⁵Cl), 450 (³⁷Cl) [M⁺+1].

To a solution of potassium tert-butoxide (865 mg; 7.08 mmol) and copper(I) chloride (23 mg; 0.24 mmol) in dimethyl formamide (4 ml) was added asolution of 4-methylthionitrobenzene (400 mg; 2.36 mmol) andmethoxyamine hydrochloride (247 mg; 2.96 mmol) in dimethyl formamide (4ml) under nitrogen. The resulting solution was heated to 40° C. for 1.5hours. The reaction mixture was then added to saturated ammoniumchloride solution (10 ml) and extracted into dichloromethane (3×10 ml).The combined organic layers were dried (magnesium sulphate), filteredand evaporated to give the crude residue, which was purified by columnchromatography (30% ethyl acetate in heptane) to give5-methylthio-2-nitroaniline (190 mg, 44%).

A solution of 5-methylthio-2-nitroaniline (50 mg; 0.27 mmol) in aceticacid (2 ml) was heated to 85° C. Zinc powder (71 mg; 1.09 mmol) was thenadded portionwise over 15 minutes and the resulting solution wasmaintained at 85° C. for 2 hours. The hot reaction mixture was thenfiltered through celite and the filter cake washed with hot acetic acid(10 ml). The filtrate was evaporated to dryness to give crude4-methylthio-1,2-phenylenediamine (42 mg, 100%). This was used directlyin the next step without purification.

Compound 11 was prepared from 4-methylthio-1,2-phenylenediamine usingroute 1: ¹H NMR (400 MHz, MeOD) δ 8.55 (s, 1H), 8.05 (d, 1H), 7.65-7.75(m, 4H), 7.6 (s, 1H), 7.5 (d, 1H), 2.65 (s, 3H), 2.55 (s, 3H); MS (ES⁺):477 (³⁵Cl), 479 (³⁷Cl) [M⁺+1].

2-(2-Chloro-5-nitrophenyl)benzimidazole (100 mg; 0.37 mmol) wasdissolved in dimethyl formamide (1 ml) and 60% sodium hydride in oil (19mg; 0.85 mmol) added. This was heated at 40° C. for 20 minutes while4-(2-chloroethyl)morpholine hydrochloride (136 mg; 0.73 mmol) wasfree-based by suspending in dimethyl formamide (1 ml) and adding 60%sodium hydride in oil (19 mg; 0.85 mmol). After 20 minutes the solutionswere combined and heated at 45° C. for 75 minutes and 85° C. for 1.5hours. The bulk of the solvent was then removed and the residuepartitioned between ethyl acetate and water. The organic phase was dried(sodium sulphate), filtered and evaporated. The residue waschromatographed on silica gel and2-(2-chloro-5-nitro-phenyl)-1-(2-morpholin-4-yl-ethyl)-1H-benzoimidazoleeluted with 40% ethyl acetate in heptane (83 mg, 58%).

The nitro group was hydrogenated using the method described for compound16 and this was then capped to give compound 12 as in route 1 step 1: ¹HNMR (400 MHz, MeOD) δ 8.15 (s, 1H), 8.1 (d, 1H), 7.85 (d, 1H), 7.75 (d,1H), 7.7 (d, 1H), 7.65 (m, 2H), 7.3-7.4 (m, 2H), 4.25-4.35 (brs, 2H),3.45-3.5 (brm, 4H), 2.7 (s, 3H), 2.6-2.7 (brm, 2H), 2.2-2.25 (brm, 4H);MS (ES⁺): 544 (³⁵Cl), 546 (³⁷Cl) [M⁺+1].

Compound 1 (50 mg; 0.12 mmol) was suspended in acetone (2 ml). 2 Mmethyl iodide in t-butyl methyl ether (70 μl; 0.14 mmol) was addedfollowed by 1 M sodium hydroxide in water (0.1 ml). The reaction wasstirred at room temperature for 1.5 hours then more base was added andstirring continued for another 2 hours. Water (3 ml) was added and themixture extracted with ethyl acetate (2×3 ml). The combined organicphases were washed with water (2×2 ml), dried (sodium sulphate),filtered and evaporated. The residue was chromatographed on silica geland compound 13 eluted with 25% ethyl acetate in heptane (21 mg, 41%).

This was converted into the HCl salt as described in route 2 step 3: ¹HNMR (400 MHz, MeOD) δ 8.4 (s, 1H), 8.1 (d, 1H), 7.95 (d, 1H), 7.9 (d,1H), 7.85 (d, 1H), 7.75 (d, 1H), 7.73 (d, 1H), 7.65-7.7 (m, 2H), 3.95(s, 3H), 2.65 (s, 3H); MS (ES⁺): 445 (³⁵Cl), 447 (³⁷Cl) [M⁺+1].

N—BOC piperidine-4-methanol (1 g; 46.5 mmol) and carbon tetrabromide(1.7 g; 51.3 mmol) were dissolved in dichloromethane (20 ml) and cooledto 0° C. Triphenyl phosphine (0.98 g; 37.3 mmol) was added and thereaction stirred at room temperature for 16 hours. Further triphenylphosphine (0.4 g; 15.7 mmol) and carbon tetrabromide (0.24 g; 7.2 mmol)were added and stirring continued for 6 hours. The solvent wasevaporated and the residue chromatographed on silica. N—BOC piperidine-4methyl bromide was eluted with 20% ethyl acetate in heptane (1.22 g,94%).

N—BOC piperidine-4 methyl bromide was converted into compound 14 usingthe method described for compound 12: ¹H NMR (400 MHz, MeOD) δ 8.2 (d,1H), 8.1 (s, 1H), 7.95 (d, 1H), 7.8 (d, 1H), 7.75 (d, 1H), 7.7-7.75 (m,2H), 7.35-7.45 (m, 2H), 4.1-4.2 (brm, 2H), 4.0 (brd, 2H), 2.75 (s, 3H),2.6-2.7 (brm, 4H), 2.0-2.15 (brm, 1H), 1.45 (s, 9H), 1-1.15 (brm, 4H);MS (ES⁺): 628 (³⁵Cl), 630 (³⁷Cl) [M⁺+1].

Compound 14 (26 mg; 0.041 mmol) was dissolved in ethanol (1 ml) andconcentrated hydrochloric acid (0.5 ml) added. After 2.5 hours stirringat room temperature, the solvent was evaporated to give compound 15 asthe dihydrochloride salt (16 mg, 73%): ¹H NMR (400 MHz, MeOD) δ 8.05 (d,1H), 7.95 (s, 1H), 7.8 (d, 1H), 7.67 (d, 1H), 7.62 (d, 1H), 7.55 (m,2H), 7.25-7.35 (m, 2H), 3.95-4.05 (brm, 2H), 2.85 (brd, 2H), 2.65 (s,3H), 2.35 (brt, 2H), 1.9 (brm, 1H), 1.3-1.4 (brm, 2H), 0.95-1.05 (brm,2H); MS (ES⁺): 528 (³⁵Cl), 530 (³⁷Cl) [M⁺+1].

1,2-diamino-4-nitrobenzene was reacted with the aldehyde from route 1step 3 using the method described in route 1 step 4 to giveN-[4-chloro-3-(5-nitro-1H-benzoimidazol-2-yl)-phenyl]-2-methyl-6-trifluoromethyl-nicotinamide.

N-[4-chloro-3-(5-nitro-1H-benzoimidazol-2-yl)-phenyl]-2-methyl-6-trifluoromethyl-nicotinamide(212 mg; 0.45 mmol) was dissolved in ethanol (4 ml) with 2 drops ofconcentrated hydrochloric acid. This was added to 5% palladium oncharcoal (42 mg; 20 wt %) and the reaction stirred under an atmosphereof hydrogen for 3 hours. The catalyst was removed by filtering throughcelite and then the solvent by evaporation to leaveN-[3-(5-amino-1H-benzoimidazol-2-yl)-4-chloro-phenyl]-2-methyl-6-trifluoromethyl-nicotinamide(198 mg, 100%).

N-[3-(5-amino-1H-benzoimidazol-2-yl)-4-chloro-phenyl]-2-methyl-6-trifluoromethyl-nicotinamide(198 mg; 0.45 mmol) was dissolved in tetrahydrofuran (2 ml) and cooledto 0° C. 60% Sodium hydride in oil (80 mg; 2.0 mmol) was added and,after 30 minutes, acetyl chloride (156 mg; 2.0 mmol). The reaction wasstirred at room temperature for 48 hours, 1.2 M hydrochloric acid (4 ml)was added and stirring continued for 4.5 hours. The mixture was basifiedwith saturated sodium bicarbonate solution, extracted with ethylacetate, dried (sodium sulphate), filtered and evaporated.Chromatography on silica gel eluting with 100% ethyl acetate gavecompound 16 (46 mg, 21%); ¹H NMR (400 MHz, MeOD) δ 8.43 (m, 1H), 8.39(s, 1H), 8.04 (d, 1H), 7.74 (d, 1H), 7.65 (m, 3H), 7.45 (d, 1H), 2.61(s, 3H) and 2.10 (s, 3H); MS (ES⁺): 488 (³⁵Cl), 490 (³⁷Cl) [M⁺+1].

Concentrated sulphuric acid (73 ml) was cooled to 0° C. before adding2-hydroxynicotinic acid (25 g; 0.18 moles) portionwise. Fuming nitricacid (17.5 ml) was added dropwise, also at 0° C., and the reaction thenheated at 50° C. for 4 hours. The mixture was cooled to room temperaturethen poured over ice/water. The resulting precipitate of2-hydroxy-5-nitronicotinic acid was filtered, washed with water anddried (29 g, 88%).

2-Hydroxy-5-nitronicotinic acid (29 g; 0.16 moles) was added portionwiseto phosphorus oxychloride (90 ml) with stirring. The temperature wasraised to 100° C. and heating continued for 3 hours before cooling toroom temperature. The bulk of the solvent was removed before adding toice-cold methanol (100 ml). The mixture was basified to pH 9 withsaturated sodium bicarbonate solution and extracted withdichloromethane. The organic phase was dried (sodium sulphate), filteredand evaporated before purification by dry flash chromatography on silicagel. Methyl 2-chloro-5-nitronicotinate was eluted with 67% ethyl acetatein heptane (31 g, 90%).

Methyl 2-chloro-5-nitronicotinate was reduced to the amine using theconditions described in route 2 step 2 and the methyl5-amino-2-chloronicotinate taken through to compound 17 using themethods described in route 1: ¹H NMR (400 MHz, MeOD) δ 9.02 (s, 1H),8.73 (s, 1H), 8.08 (d, 1H), 7.86 (m, 2H), 7.69 (d, 1H), 7.62 (d, 2H) and2.60 (s, 3H); MS (ES⁺): 432 (³⁵Cl), 434 (³⁷Cl) [M⁺+1].

Ethyl 2-aminothiazole-4-carboxylate was chlorinated as described in Tet.Lett. 2002, 43 (39), 7051-7053 (J. F. Okonya and F. Al-Obeidi). This wasthen converted to compound 18 using route 1: ¹H NMR (400 MHz, MeOD)

8.14 (d, 1H), 7.60 (d, 1H), 7.55 (m, 2H), 7.20 (m, 2H) and 2.65 (s, 3H);MS (ES⁺): 438 (³⁵Cl), 440 (³⁷Cl) [M⁺+1].

To a solution of 2-nitro-3-methoxyaniline (90 mg; 0.54 mmol) in ethanol(3 ml) was added 5% palladium on carbon (18 mg; 20 wt %). The resultingsuspension was stirred at room temperature under an atmosphere ofhydrogen for 18 hours. The suspension was then filtered through celiteand the filtrate evaporated to give 3-methoxy-1,2-phenylenediamine (73mg, 100%).

3-Methoxy-1,2-phenylenediamine was converted toN-[4-chloro-3-(7-methoxy-1H-benzoimidazol-2-yl)-phenyl]-2-methyl-6-trifluoromethyl-nicotinamideusing route 1.

A solution ofN-[4-chloro-3-(7-methoxy-1H-benzoimidazol-2-yl)-phenyl]-2-methyl-6-trifluoromethyl-nicotinamide(80 mg; 0.18 mmol) in ethanol (5 ml) was cooled to −78° C. and borontribromide (130 mg; 0.52 mmol) was added dropwise. The reaction wasallowed to warm to room temperature and stirred for 18 hours. Water (5ml) was added and the reaction mixture was basified using saturatedsodium bicarbonate solution. This was then extracted withdichloromethane (3×5 ml), the combined organic layers were dried(magnesium sulphate), filtered and evaporated to give the crude residue.This was recrystallised from hot acetonitrile to give compound 19 (10mg, 14%): ¹H NMR (400 MHz, MeOD) δ 8.15 (m, 2H), 7.95 (dd, 1H), 7.75 (d,1H), 7.65 (d, 1H), 7.15 (m, 2H), 6.7 (dd, 1H), 2.75 (s, 3H); MS (ES⁺):447 (³⁵Cl), 449 (³⁷Cl) [M⁺+1].

Compound 20 was prepared from 4-methoxy-2-nitroaniline following theprocedures described for compound 19: ¹H NMR (400 MHz, MeOD) δ 8.15 (m,2H), 7.9 (d, 1H), 7.75 (d, 1H), 7.6 (d, 1H), 7.45 (brs, 1H), 7.90 (brs,1H), 6.85 (d, 1H), 2.75 (s, 3H); MS (ES⁺): 447 (³⁵Cl), 449 (³⁷Cl)[M⁺+1].

Lithium aluminium hydride (43 mg; 1.14 mmol) was dissolved intetrahydrofuran (5 ml) and cooled to −78° C. Compound 1 (490 mg; 1.14mmol) was also dissolved in tetrahydrofuran (5 ml) and added dropwise tothe first solution. The reaction was stirred at room temperature then 1M lithium aluminium hydride in t-butyl methyl ether (2.3 ml) was addedin two portions and the reaction heated at 50° C. for 16 hours. Thereaction was quenched with water (0.13 ml), 15% sodium hydroxidesolution (0.13 ml) and water (0.39 ml) then filtered and the solidwashed with ethyl acetate. Evaporation gave compound 25 (237 mg, 50%):¹H NMR (400 MHz, MeOD) δ 7.8-7.9 (m, 3H), 7.65-7.7 (M, 2H), 7.6 (d, 1H),7.45 (d, 1H), 7.05 (s, 1H), 6.95 (d, 1H) 4.5 (s, 2H), 2.65 (s, 3H); MS(ES⁺): 417 (³⁵Cl), 419 (³⁷Cl) [M⁺+1].

4-Amino-3-nitrobenzonitrile was reduced to 4-cyano-1,2-phenylenediamineas for compound 19. This was reacted with 2-chloro-5-nitrobenzaldehydeas described in route 1 step 4 to give2-(2-chloro-5-nitro-phenyl)-1H-benzoimidazole-5-carbonitrile.

To a stirred solution of2-(2-chloro-5-nitro-phenyl)-1H-benzoimidazole-5-carbonitrile (2.00 g;6.70 mmol) in ethanol (20 ml) in a sealable tube was added triethylamine(1.90 ml; 13.4 mmol). A solution of hydroxylamine hydrochloride (933 mg;13.4 mmol) in water (4 ml) was added, the tube sealed and the mixturestirred at 75° C. for 2 hours. After cooling to room temperature, water(100 ml) was added, the precipitate filtered and washed with more waterto leave2-(2-chloro-5-nitro-phenyl)-N-hydroxy-1H-benzoimidazole-5-carboxamidine(1.76 g, 82%).

A stirred solution of2-(2-chloro-5-nitro-phenyl)-N-hydroxy-1H-benzoimidazole-5-carboxamidine(5.29 g; 16.0 mmol) in triethyl orthoformate (50 ml) was heated to 80°C. Concentrated hydrochloric acid (3 drops) was added and heatingcontinued for a further two hours. After cooling to room temperature,heptane (50 ml) was added which caused a precipitate to form. The solidwas filtered off and washed with more heptane to afford2-(2-chloro-5-nitro-phenyl)-5-[1,2,4]oxadiazol-3-yl-1H-benzoimidazole(3.30 g, 62%)

2-(2-Chloro-5-nitro-phenyl)-5-[1,2,4]oxadiazol-3-yl-1H-benzoimidazolewas converted into compound 30 using route 2 step 2 then route 1 step 1:¹H NMR (400 MHz, MeOD) δ 9.4 (s, 1H), 8.65 (d, 1H), 8.6 (s, 1H), 8.45(d, 1H), 8.15 (d, 1H), 8.05 (d, 1H), 7.75-7.85 (m, 3H), 2.75 (s, 3H); MS(ES⁺): 499 (³⁵Cl), 501 (³⁷Cl) [M⁺+1].

3,6-Dichloropyridine-2-carboxaldehyde was prepared from2,5-dichloropyridine using the procedure described in Eur. J. Org. Chem.2001, 1371-1376 (E. Marzi, A. Bigi, M. Schlosser) and converted to2-(3,6-dichloro-pyridin-2-yl)-1H-benzoimidazole using the method inroute 1 step 4.

2-(3,6-Dichloro-pyridin-2-yl)-1H-benzoimidazole (20 mg; 0.076 mmol) wasdissolved in ethanol (1.5 ml) and concentrated ammonia (2 ml) in apressure tube. After 1000 minutes in a microwave at 140° C. and 200 psi,the solvents were removed and the residue dissolved in isopropyl alcohol(1 ml) and concentrated ammonia (1 ml) before microwaving as before. Thesolvents were evaporated and the residue chromatographed on silica gelwith 80% ethyl acetate in heptane to give6-(1H-benzoimidazol-2-yl)-5-chloro-pyridin-2-ylamine (4 mg, 21%).

Compound 34 was prepared from6-(1H-benzoimidazol-2-yl)-5-chloro-pyridin-2-ylamine using the method inroute 1 step 1: ¹H NMR (400 MHz, MeOD) δ 8.55 (d, 1H), 8.2 (d, 1H), 8.1(d, 1H), 7.85 (m, 2H), 7.7 (d, 1H), 7.6 (m, 2H), 2.65 (s, 3H); MS (ES⁺):432 (³⁵Cl), 434 (³⁷Cl) [M⁺+1].

2,5-Dichloropyridine-4-carboxaldehyde was prepared from2,5-dichloropyridine using the procedure described in Eur. J. Org. Chem.2001, 1371-1376 (E. Marzi, A. Bigi, M. Schlosser) and converted to2-(2,5-dichloro-pyridin-3-yl)-1H-benzoimidazole using the method inroute 1 step 4. This was aminated as described for compound 34 and theaminopyridine converted to compound 38 using the method in route 1 step1: ¹H NMR (400 MHz, MeOD) δ 8.75 (s, 1H), 8.45 (s, 1H), 8.05 (d, 1H),7.5-7.75 (brm, 3H), 7.2-7.3 (brm, 2H), 2.65 (s, 3H); MS (ES⁺): 432(³⁵Cl), 434 (³⁷Cl) [M⁺+1].

Biological Assays Lead Compound Discovery/High-Throughput ScreeningAssay

Compounds to be tested are dissolved in DMSO to a concentration of 10mM, and stored at −20° C. To activate the Hedgehog pathway in the assaycells, an octylated (lipid-modified) form of the N-terminal fragment ofthe Sonic Hedgehog protein (OCT-SHH) is used. This N-terminal SHHfragment is produced bacterially. See, for example, Taylor F R, et al.,Biochemistry 2001, 40, 4359-71.

Compounds may be tested in the “Gli-Luc” assay below, using the cellline 10T1/2(s12), wherein the cells contain a Hedgehog-responsivereporter construct utilizing Luciferase as the reporter gene. In thisway, Hedgehog pathway signaling activity can be measured via the Gli-Lucresponse.

10t1/2(s12) cells are plated in a 96-well micro-titer plate (MTP) at20,000 cells/well in full medium [DMEM with 10% FBS]. Then plates areplaced in the incubator for incubation overnight (O/N), at 37° C. and 5%CO₂. After 24 h, the medium is replaced with Luciferase-assay medium(DMEM with 0.5% FBS). Compounds are thawed and diluted in assay mediumat 3:1000 (about 300-fold) resulting in a starting concentration ofabout 0.0003 μM to 30 μM.

Subsequently, 150 μl of each sample is added to the first wells (intriplicate). The MTP samples are then diluted at 3-fold dilutions to atotal of seven wells, ultimately resulting in a regiment of sevendilutions in triplicate, for each compound. Next, the protein ligandOCT-SHH is diluted in Luciferase-assay medium and added to each well ata final concentration of 0.3 μg/ml. Plates are then returned to theincubator for further incubation O/N, at 37° C. and 5% CO₂. After about24 h, plates are removed from the incubator and the medium isaspirated/discarded. Wells are washed once with assay buffer [PBS+1 mMMg²⁺ and 1 mM Ca²⁺]. Then 50 μl of assay buffer is added to each well.The Luciferase assay reagent is prepared as described by the vendor(LucLite kit from Packard), and 50 μl is added to each well. Plates areincubated at room temperature (RT) for about 30 minutes after which thesignals are read, again at RT, on a Topcount (Packard).

Similar assays were performed using human cell lines (specifically,human embryonic palatal mesenchyme cells, modified with the Gli-Lucconstruct as described above) in a growth medium of MEM/Sodium Pyruvatew/ 10% FBS, and an assay medium of MEM/Sodium Pyruvate w/ 0.5% FBS.OCT-SHH was added to reach a final concentration of 1 μg/ml.

Compounds identified in the above assays are depicted in Table 1.

All of the references cited above are hereby incorporated by referenceherein.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A compound represented in the general formula (I):

wherein, as valence and stability permit, X and Z, independently,represent —N(R₇)—, —O—, —S—, —(R₇)N—N(R₇)—, —ON(R₇)—, or a direct bond;Y represents —C(═O)—, —C(═S)—, —C(═NR₇)—, SO₂, or SO; A represents O, S,or NR₇; G represents a cycloalkyl, heterocyclyl, aryl, or heteroarylring fused to the ring to which it is attached; Ar represents asubstituted or unsubstituted aryl or heteroaryl ring; R₁ represents adisubstituted pyridine ring; R₂ represents from 0-4 substituents on thering to which it is attached; R₇, independently for each occurrence,represents H, lower alkyl, J-cycloalkyl, J-heterocyclyl, J-aryl,J-heteroaryl; R₈, independently for each occurrence, represents H, loweralkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; and J represents,independently for each occurrence, a chain having from 0-8 unitsselected from CK₂, NK, O, and S, wherein K represents, independently foreach occurrence, H or lower alkyl.
 2. The compound of claim 1, whereinat least one of Z and X is not a direct bond.
 3. The compound of claim1, wherein X-Y-Z includes an amide, urea, or sulfonamide.
 4. Thecompound of claim 1, wherein X is selected from —N(R₈)—, —O—, —S—, andpreferably represents NH.
 5. The compound of claim 1, wherein R₁ issubstituted with a methyl or trifluoromethyl group.
 6. The compound ofclaim 1, wherein R₁ is 6-(trifluoromethyl)-2-methylpyridin-3-yl.
 7. Thecompound of claim 1, wherein X and the ring comprising A are disposed onAr in a meta relationship.
 8. The compound of claim 1, wherein R₂represents from 1-4 substituents selected from halogen, lower alkyl,lower alkenyl, aryl, heteroaryl, carbonyl group, thiocarbonyl, ketone,aldehyde, amino, acylamino, amido, amidino, cyano, nitro, azido,sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl, sulfonamido,phosphoryl, phosphonate, phosphinate, J-R₈, J-OH, J-lower alkyl, J-loweralkenyl, J-R₈, J-SH, J-NH₂, protected forms of the above, or any two R₂,when occurring more than once in a cyclic or polycyclic structure, canbe taken together form a 4- to 8-membered cycloalkyl, aryl, orheteroaryl.
 9. The compound of claim 8, wherein R₂ represents from 1-4substituents selected from halogen, cyano, nitro, alkoxy, amino,acylamino (e.g., R₈—C(═O)NH—), a substituted or unsubstitutedcycloalkyl, heterocyclyl, aryl, or heteroaryl fused to G, andsubstituted or unsubstituted lower alkyl.
 10. A method for inhibitingactivation of a hedgehog pathway in a cell, comprising contacting thecell with a compound according to claim 1 in a sufficient amount toinhibit hedgehog signalling,
 11. The method of claim 10, wherein thecell has a phenotype of ptc loss-of-function, hedgehog gain-of-function,or smoothened gain-of-function.
 12. The method of claim 10, wherein thecompound inhibits ptc loss-of-function, hedgehog gain-of-function, orsmoothened gain-of-function mediated signal transduction with an ED₅₀ of1 μM or less.
 13. The method of claim 10, wherein the compound inhibitsptc loss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function mediated signal transduction with an ED₅₀ of 1 nM orless.
 14. The method of claim 10, wherein the cell is contacted with thehedgehog antagonist in vitro.
 15. The method of claim 10, wherein thecell is contacted with the hedgehog antagonist in vivo.
 16. The methodof claim 10, wherein the compound is administered as part of atherapeutic or cosmetic application.
 17. The method of claim 16, whereinthe therapeutic or cosmetic application is selected from regulation ofneural tissues, bone and cartilage formation and repair, regulation ofspermatogenesis, regulation of smooth muscle, regulation of lung, liverand other organs arising from the primative gut, regulation ofhematopoietic function, and regulation of skin and hair growth.
 18. Apharmaceutical preparation comprising a sterile pharmaceutical excipientand a compound according to claim
 1. 19. A method for treating orpreventing basal cell carcinoma, comprising administering thepharmaceutical preparation of claim 18 to a patient in an amountsufficient to inhibit progression of basal cell carcinoma.
 20. Acompound represented in the general formula (I):

wherein, as valence and stability permit, X and Z, independently,represent —N(R₇)—, —O—, —S—, —(R₇)N—N(R₇)—, —ON(R₇)—, or a direct bond;Y represents —C(═O)—, —C(═S)—, —C(═NR₇)—, SO₂, or SO; A represents O, S,or NR₇; G represents a cycloalkyl, heterocyclyl, aryl, or heteroarylring fused to the ring to which it is attached; Ar represents asubstituted or unsubstituted aryl or heteroaryl ring; R₁ represents apyridine ring substituted with one or more substituents, wherein atleast one of the substituents is selected from the group nitro, cyano,lower alkyl, halogenated lower alkyl, alkenyl, alkynyl, amino,alkylamino, acylamino, amido, hydroxyl, alkoxy, acyloxy, carbonyl,sulfhydryl, or sulfonyl; R₂ represents from 0-4 substituents on the ringto which it is attached; R₇, independently for each occurrence,represents H, lower alkyl, J-cycloalkyl, J-heterocyclyl, J-aryl,J-heteroaryl; R₈, independently for each occurrence, represents H, loweralkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; and J represents,independently for each occurrence, a chain having from 0-8 unitsselected from CK₂, NK, O, and S, wherein K represents, independently foreach occurrence, H or lower alkyl.
 21. The compound of claim 20, whereinat least one of Z and X is not a direct bond.
 22. The compound of claim20, wherein X-Y-Z includes an amide, urea, or sulfonamide.
 23. Thecompound of claim 20, wherein X is selected from —N(R₈)—, —O—, —S—, andpreferably represents NH.
 24. The compound of claim 20, wherein R₁ issubstituted with a methyl or trifluoromethyl group.
 25. The compound ofclaim 20, wherein R₁ is 6-(trifluoromethyl)-2-methylpyridin-3-yl. 26.The compound of claim 20, wherein X and the ring comprising A aredisposed on Ar in a meta relationship.
 27. The compound of claim 20,wherein R₂ represents from 1-4 substituents selected from halogen, loweralkyl, lower alkenyl, aryl, heteroaryl, carbonyl group, thiocarbonyl,ketone, aldehyde, amino, acylamino, amido, amidino, cyano, nitro, azido,sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl, sulfonamido,phosphoryl, phosphonate, phosphinate, J-R₈, J-OH, J-lower alkyl, J-loweralkenyl, J-R₈, J-SH, J-NH₂, protected forms of the above, or any two R₂,when occurring more than once in a cyclic or polycyclic structure, canbe taken together form a 4- to 8-membered cycloalkyl, aryl, orheteroaryl.
 28. The compound of claim 27, wherein R₂ represents from 1-4substituents selected from halogen, cyano, nitro, alkoxy, amino,acylamino (e.g., R₈—C(═O)NH—), a substituted or unsubstitutedcycloalkyl, heterocyclyl, aryl, or heteroaryl fused to G, andsubstituted or unsubstituted lower alkyl.
 29. A method for inhibitingactivation of a hedgehog pathway in a cell, comprising contacting thecell with a compound according to claim 20 in a sufficient amount toinhibit hedgehog signalling,
 30. The method of claim 29, wherein thecell has a phenotype of ptc loss-of-function, hedgehog gain-of-function,or smoothened gain-of-function.
 31. The method of claim 29, wherein thecompound inhibits ptc loss-of-function, hedgehog gain-of-function, orsmoothened gain-of-function mediated signal transduction with an ED₅₀ of1 μM or less.
 32. The method of claim 29, wherein the compound inhibitsptc loss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function mediated signal transduction with an ED₅₀ of 1 nM orless.
 33. The method of claim 29, wherein the cell is contacted with thehedgehog antagonist in vitro.
 34. The method of claim 29, wherein thecell is contacted with the hedgehog antagonist in vivo.
 35. The methodof claim 29, wherein the compound is administered as part of atherapeutic or cosmetic application.
 36. The method of claim 35, whereinthe therapeutic or cosmetic application is selected from regulation ofneural tissues, bone and cartilage formation and repair, regulation ofspermatogenesis, regulation of smooth muscle, regulation of lung, liverand other organs arising from the primative gut, regulation ofhematopoietic function, and regulation of skin and hair growth.
 37. Apharmaceutical preparation comprising a sterile pharmaceutical excipientand a compound according to claim
 20. 38. A method for treating orpreventing basal cell carcinoma, comprising administering thepharmaceutical preparation of claim 37 to a patient in an amountsufficient to inhibit progression of basal cell carcinoma.
 39. Acompound represented in the general formula (I):

wherein, as valence and stability permit, X and Z, independently,represent —N(R₇)—, —O—, —S—, —(R₇)N—N(R₇)—, —ON(R₇)—, or a direct bond;Y represents —C(═O)—, —C(═S)—, —C(═NR₇)—, SO₂, or SO; A represents O, S,or NR₇; G represents a cycloalkyl, heterocyclyl, aryl, or heteroarylring fused to the ring to which it is attached; Ar represents asubstituted or unsubstituted aryl or heteroaryl ring; R₁ represents asubstituted pyridine ring attached to J at a position meta to thenitrogen atom; R₂ represents from 0-4 substituents on the ring to whichit is attached; R₇, independently for each occurrence, represents H,lower alkyl, J-cycloalkyl, J-heterocyclyl, J-aryl, J-heteroaryl; R₈,independently for each occurrence, represents H, lower alkyl,cycloalkyl, heterocyclyl, aryl, or heteroaryl; and J represents,independently for each occurrence, a chain having from 0-8 unitsselected from CK₂, NK, O, and S, wherein K represents, independently foreach occurrence, H or lower alkyl.
 40. The compound of claim 39, whereinat least one of Z and X is not a direct bond.
 41. The compound of claim39, wherein X-Y-Z includes an amide, urea, or sulfonamide.
 42. Thecompound of claim 39, wherein X is selected from —N(R₈)—, —O—, —S—, andpreferably represents NH.
 43. The compound of claim 39, wherein R₁ issubstituted with a methyl or trifluoromethyl group.
 44. The compound ofclaim 39, wherein R₁ is 6-(trifluoromethyl)-2-methylpyridin-3-yl. 45.The compound of claim 39, wherein X and the ring comprising A aredisposed on Ar in a meta relationship.
 46. The compound of claim 39,wherein R₂ represents from 1-4 substituents selected from halogen, loweralkyl, lower alkenyl, aryl, heteroaryl, carbonyl group, thiocarbonyl,ketone, aldehyde, amino, acylamino, amido, amidino, cyano, nitro, azido,sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl, sulfonamido,phosphoryl, phosphonate, phosphinate, J-R₈, J-OH, J-lower alkyl, J-loweralkenyl, J-R₈, J-SH, J-NH₂, protected forms of the above, or any two R₂,when occurring more than once in a cyclic or polycyclic structure, canbe taken together form a 4- to 8-membered cycloalkyl, aryl, orheteroaryl.
 47. The compound of claim 46, wherein R₂ represents from 1-4substituents selected from halogen, cyano, nitro, alkoxy, amino,acylamino (e.g., R₈—C(═O)NH—), a substituted or unsubstitutedcycloalkyl, heterocyclyl, aryl, or heteroaryl fused to G, andsubstituted or unsubstituted lower alkyl.
 48. A method for inhibitingactivation of a hedgehog pathway in a cell, comprising contacting thecell with a compound according to claim 1 in a sufficient amount toinhibit hedgehog signalling,
 49. The method of claim 48, wherein thecell has a phenotype of ptc loss-of-function, hedgehog gain-of-function,or smoothened gain-of-function.
 50. The method of claim 48, wherein thecompound inhibits ptc loss-of-function, hedgehog gain-of-function, orsmoothened gain-of-function mediated signal transduction with an ED₅₀ of1 μM or less.
 51. The method of claim 48, wherein the compound inhibitsptc loss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function mediated signal transduction with an ED₅₀ of 1 nM orless.
 52. The method of claim 48, wherein the cell is contacted with thehedgehog antagonist in vitro.
 53. The method of claim 48, wherein thecell is contacted with the hedgehog antagonist in vivo.
 54. The methodof claim 48, wherein the compound is administered as part of atherapeutic or cosmetic application.
 55. The method of claim 54, whereinthe therapeutic or cosmetic application is selected from regulation ofneural tissues, bone and cartilage formation and repair, regulation ofspermatogenesis, regulation of smooth muscle, regulation of lung, liverand other organs arising from the primative gut, regulation ofhematopoietic function, and regulation of skin and hair growth.
 56. Apharmaceutical preparation comprising a sterile pharmaceutical excipientand a compound according to claim
 39. 57. A method for treating orpreventing basal cell carcinoma, comprising administering thepharmaceutical preparation of claim 56 to a patient in an amountsufficient to inhibit progression of basal cell carcinoma.